Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

Directory of Open Access Journals (Sweden)

Borges-Pérez Andrés

2008-12-01

Full Text Available Abstract Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC, SGN-U321250 (TIP41, SGN-U346908 ("Expressed" and SGN-U316474 (SAND genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

Directory of Open Access Journals (Sweden)

Pek-Lan Chan

Full Text Available BACKGROUND: The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR. With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. RESULTS: In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569 outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN. PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. CONCLUSIONS: Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection

Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

Science.gov (United States)

Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate

Real time quantitative amplification detection on a microarray: towards high multiplex quantitative PCR.

NARCIS (Netherlands)

Pierik, A.; Moamfa, M; van Zelst, M.; Clout, D.; Stapert, H.; Dijksman, Johan Frederik; Broer, D.; Wimberger-Friedl, R.

2012-01-01

Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that

Real time quantitative amplification detection on a microarray : towards high multiplex quantitative PCR

NARCIS (Netherlands)

Pierik, Anke; Boamfa, M.; Zelst, van M.; Clout, D.; Stapert, H.R.; Dijksman, J.F.; Broer, D.J.; Wimberger-Friedl, R.

2012-01-01

Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that

A new ChainMail approach for real-time soft tissue simulation.

Science.gov (United States)

Zhang, Jinao; Zhong, Yongmin; Smith, Julian; Gu, Chengfan

2016-07-03

This paper presents a new ChainMail method for real-time soft tissue simulation. This method enables the use of different material properties for chain elements to accommodate various materials. Based on the ChainMail bounding region, a new time-saving scheme is developed to improve computational efficiency for isotropic materials. The proposed method also conserves volume and strain energy. Experimental results demonstrate that the proposed ChainMail method can not only accommodate isotropic, anisotropic and heterogeneous materials but also model incompressibility and relaxation behaviors of soft tissues. Further, the proposed method can achieve real-time computational performance.

Real-time haptic cutting of high-resolution soft tissues.

Science.gov (United States)

Wu, Jun; Westermann, Rüdiger; Dick, Christian

2014-01-01

We present our systematic efforts in advancing the computational performance of physically accurate soft tissue cutting simulation, which is at the core of surgery simulators in general. We demonstrate a real-time performance of 15 simulation frames per second for haptic soft tissue cutting of a deformable body at an effective resolution of 170,000 finite elements. This is achieved by the following innovative components: (1) a linked octree discretization of the deformable body, which allows for fast and robust topological modifications of the simulation domain, (2) a composite finite element formulation, which thoroughly reduces the number of simulation degrees of freedom and thus enables to carefully balance simulation performance and accuracy, (3) a highly efficient geometric multigrid solver for solving the linear systems of equations arising from implicit time integration, (4) an efficient collision detection algorithm that effectively exploits the composition structure, and (5) a stable haptic rendering algorithm for computing the feedback forces. Considering that our method increases the finite element resolution for physically accurate real-time soft tissue cutting simulation by an order of magnitude, our technique has a high potential to significantly advance the realism of surgery simulators.

Quantitative density measurements from a real-time neutron radiography system

International Nuclear Information System (INIS)

McRae, D.D.; Jenkins, R.W. Jr.; Brenizer, J.S.; Tobin, K.W.; Hosticka, B.; Sulcoski, M.F.

1986-01-01

An advanced video system has been assembled from commercially available equipment to support the real-time neutron radiography facility established jointly by the University of Virginia Department of Nuclear Engineering and Engineering Physics, and the Philip Morris Research Center. A schematic diagram of the equipment used for real-time neutron radiography is presented. To obtain quantitative density measurements with this system, several modifications of both hardware and image processing software were required. After implementation of these changes, the system was capable of determining material densities by measuring the degree of neutron attenuation

Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

DEFF Research Database (Denmark)

Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

2004-01-01

We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods...

[Real-time PCR in rapid diagnosis of Aeromonas hydrophila necrotizing soft tissue infections].

Science.gov (United States)

Kohayagawa, Yoshitaka; Izumi, Yoko; Ushita, Misuzu; Niinou, Norio; Koshizaki, Masayuki; Yamamori, Yuji; Kaneko, Sakae; Fukushima, Hiroshi

2009-11-01

We report a case of rapidly progressive necrotizing soft tissue infection and sepsis followed by a patient's death. We suspected Vibrio vulnificus infection because the patient's underlying disease was cirrhosis and the course extremely rapid. No microbe had been detected at death. We extracted DNA from a blood culture bottle. SYBR green I real-time PCR was conducted but could not detect V. vulnificus vvh in the DNA sample. Aeromonas hydrophila was cultured and identified in blood and necrotized tissue samples. Real-time PCR was conducted to detect A. hydrophila ahh1, AHCYTOEN and aerA in the DNA sample extracted from the blood culture bottle and an isolated necrotized tissue strain, but only ahh1 was positive. High-mortality in necrotizing soft tissue infections makes it is crucial to quickly detect V. vulnificus and A. hydrophila. We found real-time PCR for vvh, ahh1, AHCYTOEN, and aerA useful in detecting V. vulnificus and A. hydrophila in necrotizing soft tissue infections.

Real-time quantitative phase reconstruction in off-axis digital holography using multiplexing.

Science.gov (United States)

Girshovitz, Pinhas; Shaked, Natan T

2014-04-15

We present a new approach for obtaining significant speedup in the digital processing of extracting unwrapped phase profiles from off-axis digital holograms. The new technique digitally multiplexes two orthogonal off-axis holograms, where the digital reconstruction, including spatial filtering and two-dimensional phase unwrapping on a decreased number of pixels, can be performed on both holograms together, without redundant operations. Using this technique, we were able to reconstruct, for the first time to our knowledge, unwrapped phase profiles from off-axis holograms with 1 megapixel in more than 30 frames per second using a standard single-core personal computer on a MATLAB platform, without using graphic-processing-unit programming or parallel computing. This new technique is important for real-time quantitative visualization and measurements of highly dynamic samples and is applicable for a wide range of applications, including rapid biological cell imaging and real-time nondestructive testing. After comparing the speedups obtained by the new technique for holograms of various sizes, we present experimental results of real-time quantitative phase visualization of cells flowing rapidly through a microchannel.

Quantitative real-time RT-PCR and chromogenic in situ hybridization

DEFF Research Database (Denmark)

Rosa, Fabíola E; Silveira, Sara M; Silveira, Cássia G T

2009-01-01

. METHODS: To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. RESULTS: The concordance...

Application of 42K to Arabidopsis tissues using real-time radioisotope imaging system (RRIS)

International Nuclear Information System (INIS)

Aramaki, Toshinori; Sugita, Ryohei; Hirose, Atsushi; Kobayashi, Natsuko I.; Tanoi, Keitaro; Nakanishi, Tomoko M.

2015-01-01

We performed an imaging analysis of 42 K in Arabidopsis using real-time radioisotope imaging system (RRIS). First, we purified 42 K from an 42 Ar - 42 K generator. And then, we characterized RRIS performance by quantitatively determining 42 K using standard series. As a result, the dynamic range for 42 K was determined to be at least three orders of magnitude. Next, we evaluated the level of self-absorption in Arabidopsis organs by comparing the signal intensity detected using RRIS and the actual radioactivity detected by a gamma-counting method. There was no significant difference in detection efficiency between the thick bolt(stem) tissue and the thin leaf tissue. The reason for scarce self-absorption could be related to the relatively strong β ray emissions that have a maximum energy of 3525.4 keV. (author)

Real time quantitative phase microscopy based on single-shot transport of intensity equation (ssTIE) method

Science.gov (United States)

Yu, Wei; Tian, Xiaolin; He, Xiaoliang; Song, Xiaojun; Xue, Liang; Liu, Cheng; Wang, Shouyu

2016-08-01

Microscopy based on transport of intensity equation provides quantitative phase distributions which opens another perspective for cellular observations. However, it requires multi-focal image capturing while mechanical and electrical scanning limits its real time capacity in sample detections. Here, in order to break through this restriction, real time quantitative phase microscopy based on single-shot transport of the intensity equation method is proposed. A programmed phase mask is designed to realize simultaneous multi-focal image recording without any scanning; thus, phase distributions can be quantitatively retrieved in real time. It is believed the proposed method can be potentially applied in various biological and medical applications, especially for live cell imaging.

Real-time soft tissue motion estimation for lung tumors during radiotherapy delivery.

Science.gov (United States)

Rottmann, Joerg; Keall, Paul; Berbeco, Ross

2013-09-01

To provide real-time lung tumor motion estimation during radiotherapy treatment delivery without the need for implanted fiducial markers or additional imaging dose to the patient. 2D radiographs from the therapy beam's-eye-view (BEV) perspective are captured at a frame rate of 12.8 Hz with a frame grabber allowing direct RAM access to the image buffer. An in-house developed real-time soft tissue localization algorithm is utilized to calculate soft tissue displacement from these images in real-time. The system is tested with a Varian TX linear accelerator and an AS-1000 amorphous silicon electronic portal imaging device operating at a resolution of 512 × 384 pixels. The accuracy of the motion estimation is verified with a dynamic motion phantom. Clinical accuracy was tested on lung SBRT images acquired at 2 fps. Real-time lung tumor motion estimation from BEV images without fiducial markers is successfully demonstrated. For the phantom study, a mean tracking error real-time markerless lung tumor motion estimation from BEV images alone. The described system can operate at a frame rate of 12.8 Hz and does not require prior knowledge to establish traceable landmarks for tracking on the fly. The authors show that the geometric accuracy is similar to (or better than) previously published markerless algorithms not operating in real-time.

Real-time simulation of the nonlinear visco-elastic deformations of soft tissues.

Science.gov (United States)

Basafa, Ehsan; Farahmand, Farzam

2011-05-01

Mass-spring-damper (MSD) models are often used for real-time surgery simulation due to their fast response and fairly realistic deformation replication. An improved real time simulation model of soft tissue deformation due to a laparoscopic surgical indenter was developed and tested. The mechanical realization of conventional MSD models was improved using nonlinear springs and nodal dampers, while their high computational efficiency was maintained using an adapted implicit integration algorithm. New practical algorithms for model parameter tuning, collision detection, and simulation were incorporated. The model was able to replicate complex biological soft tissue mechanical properties under large deformations, i.e., the nonlinear and viscoelastic behaviors. The simulated response of the model after tuning of its parameters to the experimental data of a deer liver sample, closely tracked the reference data with high correlation and maximum relative differences of less than 5 and 10%, for the tuning and testing data sets respectively. Finally, implementation of the proposed model and algorithms in a graphical environment resulted in a real-time simulation with update rates of 150 Hz for interactive deformation and haptic manipulation, and 30 Hz for visual rendering. The proposed real time simulation model of soft tissue deformation due to a laparoscopic surgical indenter was efficient, realistic, and accurate in ex vivo testing. This model is a suitable candidate for testing in vivo during laparoscopic surgery.

Progesterone receptor isoform analysis by quantitative real-time polymerase chain reaction in formalin-fixed, paraffin-embedded canine mammary dysplasias and tumors

DEFF Research Database (Denmark)

Guil-Luna, S.; Stenvang, Jan; Brünner, Nils

2014-01-01

and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign...... in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors...

Real-time and quantitative isotropic spatial resolution susceptibility imaging for magnetic nanoparticles

Science.gov (United States)

Pi, Shiqiang; Liu, Wenzhong; Jiang, Tao

2018-03-01

The magnetic transparency of biological tissue allows the magnetic nanoparticle (MNP) to be a promising functional sensor and contrast agent. The complex susceptibility of MNPs, strongly influenced by particle concentration, excitation magnetic field and their surrounding microenvironment, provides significant implications for biomedical applications. Therefore, magnetic susceptibility imaging of high spatial resolution will give more detailed information during the process of MNP-aided diagnosis and therapy. In this study, we present a novel spatial magnetic susceptibility extraction method for MNPs under a gradient magnetic field, a low-frequency drive magnetic field, and a weak strength high-frequency magnetic field. Based on this novel method, a magnetic particle susceptibility imaging (MPSI) of millimeter-level spatial resolution (<3 mm) was achieved using our homemade imaging system. Corroborated by the experimental results, the MPSI shows real-time (1 s per frame acquisition) and quantitative abilities, and isotropic high resolution.

Quantitative real-time monitoring of dryer effluent using fiber optic near-infrared spectroscopy.

Science.gov (United States)

Harris, S C; Walker, D S

2000-09-01

This paper describes a method for real-time quantitation of the solvents evaporating from a dryer. The vapor stream in the vacuum line of a dryer was monitored in real time using a fiber optic-coupled acousto-optic tunable filter near-infrared (AOTF-NIR) spectrometer. A balance was placed in the dryer, and mass readings were recorded for every scan of the AOTF-NIR. A partial least-squares (PLS) calibration was subsequently built based on change in mass over change in time for solvents typically used in a chemical manufacturing plant. Controlling software for the AOTF-NIR was developed. The software collects spectra, builds the PLS calibration model, and continuously fits subsequently collected spectra to the calibration, allowing the operator to follow the mass loss of solvent from the dryer. The results indicate that solvent loss can be monitored and quantitated in real time using NIR for the optimization of drying times. These time-based mass loss values have also been used to calculate "dynamic" vapor density values for the solvents. The values calculated are in agreement with values determined from the ideal gas law and could prove valuable as tools to measure temperature or pressure indirectly.

Identification of Reference Genes for Normalizing Quantitative Real-Time PCR in Urechis unicinctus

Science.gov (United States)

Bai, Yajiao; Zhou, Di; Wei, Maokai; Xie, Yueyang; Gao, Beibei; Qin, Zhenkui; Zhang, Zhifeng

2018-06-01

The reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most important techniques of studying gene expression. A set of valid reference genes are essential for the accurate normalization of data. In this study, five candidate genes were analyzed with geNorm, NormFinder, BestKeeper and ΔCt methods to identify the genes stably expressed in echiuran Urechis unicinctus, an important commercial marine benthic worm, under abiotic (sulfide stress) and normal (adult tissues, embryos and larvae at different development stages) conditions. The comprehensive results indicated that the expression of TBP was the most stable at sulfide stress and in developmental process, while the expression of EF- 1- α was the most stable at sulfide stress and in various tissues. TBP and EF- 1- α were recommended as a suitable reference gene combination to accurately normalize the expression of target genes at sulfide stress; and EF- 1- α, TBP and TUB were considered as a potential reference gene combination for normalizing the expression of target genes in different tissues. No suitable gene combination was obtained among these five candidate genes for normalizing the expression of target genes for developmental process of U. unicinctus. Our results provided a valuable support for quantifying gene expression using RT-qPCR in U. unicinctus.

Real-time soft tissue motion estimation for lung tumors during radiotherapy delivery

International Nuclear Information System (INIS)

Rottmann, Joerg; Berbeco, Ross; Keall, Paul

2013-01-01

Purpose: To provide real-time lung tumor motion estimation during radiotherapy treatment delivery without the need for implanted fiducial markers or additional imaging dose to the patient.Methods: 2D radiographs from the therapy beam's-eye-view (BEV) perspective are captured at a frame rate of 12.8 Hz with a frame grabber allowing direct RAM access to the image buffer. An in-house developed real-time soft tissue localization algorithm is utilized to calculate soft tissue displacement from these images in real-time. The system is tested with a Varian TX linear accelerator and an AS-1000 amorphous silicon electronic portal imaging device operating at a resolution of 512 × 384 pixels. The accuracy of the motion estimation is verified with a dynamic motion phantom. Clinical accuracy was tested on lung SBRT images acquired at 2 fps.Results: Real-time lung tumor motion estimation from BEV images without fiducial markers is successfully demonstrated. For the phantom study, a mean tracking error <1.0 mm [root mean square (rms) error of 0.3 mm] was observed. The tracking rms accuracy on BEV images from a lung SBRT patient (≈20 mm tumor motion range) is 1.0 mm.Conclusions: The authors demonstrate for the first time real-time markerless lung tumor motion estimation from BEV images alone. The described system can operate at a frame rate of 12.8 Hz and does not require prior knowledge to establish traceable landmarks for tracking on the fly. The authors show that the geometric accuracy is similar to (or better than) previously published markerless algorithms not operating in real-time

Real-time soft tissue motion estimation for lung tumors during radiotherapy delivery

Energy Technology Data Exchange (ETDEWEB)

Rottmann, Joerg; Berbeco, Ross [Brigham and Women' s Hospital, Dana Farber-Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115 (United States); Keall, Paul [Radiation Physics Laboratory, Sydney Medical School, University of Sydney, Sydney NSW 2006 (Australia)

2013-09-15

Purpose: To provide real-time lung tumor motion estimation during radiotherapy treatment delivery without the need for implanted fiducial markers or additional imaging dose to the patient.Methods: 2D radiographs from the therapy beam's-eye-view (BEV) perspective are captured at a frame rate of 12.8 Hz with a frame grabber allowing direct RAM access to the image buffer. An in-house developed real-time soft tissue localization algorithm is utilized to calculate soft tissue displacement from these images in real-time. The system is tested with a Varian TX linear accelerator and an AS-1000 amorphous silicon electronic portal imaging device operating at a resolution of 512 × 384 pixels. The accuracy of the motion estimation is verified with a dynamic motion phantom. Clinical accuracy was tested on lung SBRT images acquired at 2 fps.Results: Real-time lung tumor motion estimation from BEV images without fiducial markers is successfully demonstrated. For the phantom study, a mean tracking error <1.0 mm [root mean square (rms) error of 0.3 mm] was observed. The tracking rms accuracy on BEV images from a lung SBRT patient (≈20 mm tumor motion range) is 1.0 mm.Conclusions: The authors demonstrate for the first time real-time markerless lung tumor motion estimation from BEV images alone. The described system can operate at a frame rate of 12.8 Hz and does not require prior knowledge to establish traceable landmarks for tracking on the fly. The authors show that the geometric accuracy is similar to (or better than) previously published markerless algorithms not operating in real-time.

Expression analysis of fusarium wilt resistance gene in melon by real-time quantitative pcr

International Nuclear Information System (INIS)

Wang, X.; Xu, B.; Zhao, L.; Gao, P.; Luan, F.

2014-01-01

Melon Actin gene was used as a reference gene, to explore the gene expression profiles of the Fom-2 gene in roots, stems, and leaves of melon MR-1 under induction by Fusarium oxysporum f. sp. melonis. Monitoring using real-time quantitative PCR showed similar accumulation patterns of Fom-2 in roots, stems, and leaves over the observation period of 1 to 11 days; the expression level in stems was the highest. The expression of the Fom-2 gene was strengthened by the prolongation of induction time. In stems, the expression of Fom-2 was 5.737 times higher than in the control at three days; in roots, expression of Fom-2 was 5.617 times higher than in the control at five days. Similarly, the expression of Fom-2 in leaves obviously increased. It was 4.441 times higher than in the control at 5 days. The expression of Fom-2 was non-tissue specific, up-regulated under induction by Fusarium, and related to early resistance to Fusarium wilt. (author)

Evaluation of human muscle hardness after dynamic exercise with ultrasound real-time tissue elastography: A feasibility study

Energy Technology Data Exchange (ETDEWEB)

Yanagisawa, O., E-mail: o.yanagisawa@aoni.waseda.jp [Faculty of Sport Sciences, Waseda University, Tokorozawa, Saitama (Japan); Niitsu, M. [Department of Radiological Science, Faculty of Health Sciences, Tokyo Metropolitan University, Arakawa-ku, Tokyo (Japan); Kurihara, T. [Faculty of Sport and Health Science, Ritsumeikan University, Kusatsu, Shiga (Japan); Fukubayashi, T. [Faculty of Sport Sciences, Waseda University, Tokorozawa, Saitama (Japan)

2011-09-15

Aim: To assess the feasibility of ultrasound real-time tissue elastography (RTE) for measuring exercise-induced changes in muscle hardness and to compare the findings of RTE with those of a tissue hardness meter for semi-quantitative assessment of the hardness of exercised muscles. Materials and methods: Nine male participants performed an arm-curl exercise. RTE measurements were performed by manually applying repetitive compression with the transducer on the scan position before exercise, immediately after exercise, and at 30 min after exercise; strain ratios between muscle and a reference material (hydrogel) were calculated (muscle strain/material strain). A tissue hardness meter was also used to evaluate muscle hardness. The intraclass correlation coefficients (ICCs) for the three repeated measurements at each measurement time were calculated to evaluate the intra-observer reproducibility of each technique. Results: Immediately after exercise, the strain ratio and the value obtained using the tissue hardness meter significantly decreased (from 1.65 to 1.35) and increased (from 51.8 to 54.3), respectively. Both parameters returned to their pre-exercise value 30 min after exercise. The ICCs of the RTE (and the ICCs of the muscle hardness meter) were 0.971 (0.816) before exercise, 0.939 (0.776) immediately after exercise, and 0.959 (0.882) at 30 min after exercise. Conclusion: Similar to the muscle hardness meter, RTE revealed the exercise-induced changes of muscle hardness semi-quantitatively. The intra-observer reproducibility of RTE was very high at each measurement time. These findings suggest that RTE is a clinically useful technique for assessing hardness of specific exercised muscles.

Evaluation of human muscle hardness after dynamic exercise with ultrasound real-time tissue elastography: A feasibility study

International Nuclear Information System (INIS)

Yanagisawa, O.; Niitsu, M.; Kurihara, T.; Fukubayashi, T.

2011-01-01

Aim: To assess the feasibility of ultrasound real-time tissue elastography (RTE) for measuring exercise-induced changes in muscle hardness and to compare the findings of RTE with those of a tissue hardness meter for semi-quantitative assessment of the hardness of exercised muscles. Materials and methods: Nine male participants performed an arm-curl exercise. RTE measurements were performed by manually applying repetitive compression with the transducer on the scan position before exercise, immediately after exercise, and at 30 min after exercise; strain ratios between muscle and a reference material (hydrogel) were calculated (muscle strain/material strain). A tissue hardness meter was also used to evaluate muscle hardness. The intraclass correlation coefficients (ICCs) for the three repeated measurements at each measurement time were calculated to evaluate the intra-observer reproducibility of each technique. Results: Immediately after exercise, the strain ratio and the value obtained using the tissue hardness meter significantly decreased (from 1.65 to 1.35) and increased (from 51.8 to 54.3), respectively. Both parameters returned to their pre-exercise value 30 min after exercise. The ICCs of the RTE (and the ICCs of the muscle hardness meter) were 0.971 (0.816) before exercise, 0.939 (0.776) immediately after exercise, and 0.959 (0.882) at 30 min after exercise. Conclusion: Similar to the muscle hardness meter, RTE revealed the exercise-induced changes of muscle hardness semi-quantitatively. The intra-observer reproducibility of RTE was very high at each measurement time. These findings suggest that RTE is a clinically useful technique for assessing hardness of specific exercised muscles.

Real time assessment of RF cardiac tissue ablation with optical spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Demos, S G; Sharareh, S

2008-03-20

An optical spectroscopy approach is demonstrated allowing for critical parameters during RF ablation of cardiac tissue to be evaluated in real time. The method is based on incorporating in a typical ablation catheter transmitting and receiving fibers that terminate at the tip of the catheter. By analyzing the spectral characteristics of the NIR diffusely reflected light, information is obtained on such parameters as, catheter-tissue proximity, lesion formation, depth of penetration of the lesion, formation of char during the ablation, formation of coagulum around the ablation site, differentiation of ablated from healthy tissue, and recognition of micro-bubble formation in the tissue.

Intra-laboratory validation of chronic bee paralysis virus quantitation using an accredited standardised real-time quantitative RT-PCR method.

Science.gov (United States)

Blanchard, Philippe; Regnault, Julie; Schurr, Frank; Dubois, Eric; Ribière, Magali

2012-03-01

Chronic bee paralysis virus (CBPV) is responsible for chronic bee paralysis, an infectious and contagious disease in adult honey bees (Apis mellifera L.). A real-time RT-PCR assay to quantitate the CBPV load is now available. To propose this assay as a reference method, it was characterised further in an intra-laboratory study during which the reliability and the repeatability of results and the performance of the assay were confirmed. The qPCR assay alone and the whole quantitation method (from sample RNA extraction to analysis) were both assessed following the ISO/IEC 17025 standard and the recent XP U47-600 standard issued by the French Standards Institute. The performance of the qPCR assay and of the overall CBPV quantitation method were validated over a 6 log range from 10(2) to 10(8) with a detection limit of 50 and 100 CBPV RNA copies, respectively, and the protocol of the real-time RT-qPCR assay for CBPV quantitation was approved by the French Accreditation Committee. Copyright © 2011 Elsevier B.V. All rights reserved.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

Science.gov (United States)

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-11-01

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

Tissue-specific Calibration of Real-time PCR Facilitates Absolute Quantification of Plasmid DNA in Biodistribution Studies

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Joan K Ho

2016-01-01

Full Text Available Analysis of the tissue distribution of plasmid DNA after administration of nonviral gene delivery systems is best accomplished using quantitative real-time polymerase chain reaction (qPCR, although published strategies do not allow determination of the absolute mass of plasmid delivered to different tissues. Generally, data is expressed as the mass of plasmid relative to the mass of genomic DNA (gDNA in the sample. This strategy is adequate for comparisons of efficiency of delivery to a single site but it does not allow direct comparison of delivery to multiple tissues, as the mass of gDNA extracted per unit mass of each tissue is different. We show here that by constructing qPCR standard curves for each tissue it is possible to determine the dose of intact plasmid remaining in each tissue, which is a more useful parameter when comparing the fates of different formulations of DNA. We exemplify the use of this tissue-specific qPCR method by comparing the delivery of naked DNA, cationic DNA complexes, and neutral PEGylated DNA complexes after intramuscular injection. Generally, larger masses of intact plasmid were present 24 hours after injection of DNA complexes, and neutral complexes resulted in delivery of a larger mass of intact plasmid to the spleen.

Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction.

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Zornhagen, K W; Kristensen, A T; Hansen, A E; Oxboel, J; Kjaer, A

2015-12-01

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours. The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm was used to analyse the most suitable reference genes. Eight potential reference genes were excluded from this final analysis because of their dissociation curves. β-Glucuronidase (GUSB) and proteasome subunit, beta type, 6 (PSMB6) were most stably expressed with an M value of 0.154 and a CV of 0.053 describing their average stability. We suggest that choice of reference genes should be based on specific testing in every new experimental set-up. © 2014 John Wiley & Sons Ltd.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

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Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-12-14

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

Real-time simulation of contact and cutting of heterogeneous soft-tissues.

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Courtecuisse, Hadrien; Allard, Jérémie; Kerfriden, Pierre; Bordas, Stéphane P A; Cotin, Stéphane; Duriez, Christian

2014-02-01

This paper presents a numerical method for interactive (real-time) simulations, which considerably improves the accuracy of the response of heterogeneous soft-tissue models undergoing contact, cutting and other topological changes. We provide an integrated methodology able to deal both with the ill-conditioning issues associated with material heterogeneities, contact boundary conditions which are one of the main sources of inaccuracies, and cutting which is one of the most challenging issues in interactive simulations. Our approach is based on an implicit time integration of a non-linear finite element model. To enable real-time computations, we propose a new preconditioning technique, based on an asynchronous update at low frequency. The preconditioner is not only used to improve the computation of the deformation of the tissues, but also to simulate the contact response of homogeneous and heterogeneous bodies with the same accuracy. We also address the problem of cutting the heterogeneous structures and propose a method to update the preconditioner according to the topological modifications. Finally, we apply our approach to three challenging demonstrators: (i) a simulation of cataract surgery (ii) a simulation of laparoscopic hepatectomy (iii) a brain tumor surgery. Copyright © 2013 Elsevier B.V. All rights reserved.

Detection of medically important Candida species by absolute quantitation real-time polymerase chain reaction.

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Than, Leslie Thian Lung; Chong, Pei Pei; Ng, Kee Peng; Seow, Heng Fong

2015-01-01

The number of invasive candidiasis cases has risen especially with an increase in the number of immunosuppressed and immunocom promised patients. The early detection of Candida species which is specific and sensitive is important in determining the correct administration of antifungal drugs to patients. This study aims to develop a method for the detection, identification and quantitation of medically important Candida species through quantitative polymerase chain reaction (qPCR). The isocitrate lyase (ICL) gene which is not found in mammals was chosen as the target gene of real-time PCR. Absolute quantitation of the gene copy number was achieved by constructing the plasmid containing the ICL gene which is used to generate standard curve. Twenty fungal species, two bacterial species and human DNA were tested to check the specificity of the detection method. All eight Candida species were successfully detected, identified and quantitated based on the ICL gene. A seven-log range of the gene copy number and a minimum detection limit of 10(3) copies were achieved. A one-tube absolute quantification real-time PCR that differentiates medically important Candida species via individual unique melting temperature was achieved. Analytical sensitivity and specificity were not compromised.

Statistical aspects of quantitative real-time PCR experiment design.

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Kitchen, Robert R; Kubista, Mikael; Tichopad, Ales

2010-04-01

Experiments using quantitative real-time PCR to test hypotheses are limited by technical and biological variability; we seek to minimise sources of confounding variability through optimum use of biological and technical replicates. The quality of an experiment design is commonly assessed by calculating its prospective power. Such calculations rely on knowledge of the expected variances of the measurements of each group of samples and the magnitude of the treatment effect; the estimation of which is often uninformed and unreliable. Here we introduce a method that exploits a small pilot study to estimate the biological and technical variances in order to improve the design of a subsequent large experiment. We measure the variance contributions at several 'levels' of the experiment design and provide a means of using this information to predict both the total variance and the prospective power of the assay. A validation of the method is provided through a variance analysis of representative genes in several bovine tissue-types. We also discuss the effect of normalisation to a reference gene in terms of the measured variance components of the gene of interest. Finally, we describe a software implementation of these methods, powerNest, that gives the user the opportunity to input data from a pilot study and interactively modify the design of the assay. The software automatically calculates expected variances, statistical power, and optimal design of the larger experiment. powerNest enables the researcher to minimise the total confounding variance and maximise prospective power for a specified maximum cost for the large study. Copyright 2010 Elsevier Inc. All rights reserved.

A novel mouse model of soft-tissue infection using bioluminescence imaging allows noninvasive, real-time monitoring of bacterial growth.

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Yoshioka, Kenji; Ishii, Ken; Kuramoto, Tetsuya; Nagai, Shigenori; Funao, Haruki; Ishihama, Hiroko; Shiono, Yuta; Sasaki, Aya; Aizawa, Mamoru; Okada, Yasunori; Koyasu, Shigeo; Toyama, Yoshiaki; Matsumoto, Morio

2014-01-01

Musculoskeletal infections, including surgical-site and implant-associated infections, often cause progressive inflammation and destroy areas of the soft tissue. Treating infections, especially those caused by multi-antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) remains a challenge. Although there are a few animal models that enable the quantitative evaluation of infection in soft tissues, these models are not always reproducible or sustainable. Here, we successfully established a real-time, in vivo, quantitative mouse model of soft-tissue infection in the superficial gluteus muscle (SGM) using bioluminescence imaging. A bioluminescent strain of MRSA was inoculated into the SGM of BALB/c adult male mice, followed by sequential measurement of bacterial photon intensity and serological and histological analyses of the mice. The mean photon intensity in the mice peaked immediately after inoculation and remained stable until day 28. The serum levels of interleukin-6, interleukin-1 and C-reactive protein at 12 hours after inoculation were significantly higher than those prior to inoculation, and the C-reactive protein remained significantly elevated until day 21. Histological analyses showed marked neutrophil infiltration and abscesses containing necrotic and fibrous tissues in the SGM. With this SGM mouse model, we successfully visualized and quantified stable bacterial growth over an extended period of time with bioluminescence imaging, which allowed us to monitor the process of infection without euthanizing the experimental animals. This model is applicable to in vivo evaluations of the long-term efficacy of novel antibiotics or antibacterial implants.

Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

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Rueda-Martínez, Carmen; Fernández, M Carmen; Soto-Navarrete, María Teresa; Jiménez-Navarro, Manuel; Durán, Ana Carmen; Fernández, Borja

2016-01-01

Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180-240 days old) and 56 old (300-440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

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Carmen Rueda-Martínez

Full Text Available Bicuspid aortic valve (BAV is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40% incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR assays. A total of 51 adult (180-240 days old and 56 old (300-440 days old animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30, or to the affected strain of hamsters with TAV (n = 45 or BAV (n = 32. The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

Noninvasive Real-Time Assessment of Cell Viability in a Three-Dimensional Tissue.

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Mahfouzi, Seyed Hossein; Amoabediny, Ghassem; Doryab, Ali; Safiabadi-Tali, Seyed Hamid; Ghanei, Mostafa

2018-04-01

Maintaining cell viability within 3D tissue engineering scaffolds is an essential step toward a functional tissue or organ. Assessment of cell viability in 3D scaffolds is necessary to control and optimize tissue culture process. Monitoring systems based on respiration activity of cells (e.g., oxygen consumption) have been used in various cell cultures. In this research, an online monitoring system based on respiration activity was developed to monitor cell viability within acellular lung scaffolds. First, acellular lung scaffolds were recellularized with human umbilical cord vein endothelial cells, and then, cell viability was monitored during a 5-day period. The real-time monitoring system generated a cell growth profile representing invaluable information on cell viability and proliferative states during the culture period. The cell growth profile obtained by the monitoring system was consistent with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and glucose consumption measurement. This system provided a means for noninvasive, real-time, and repetitive investigation of cell viability. Also, we showed the applicability of this monitoring system by introducing shaking as an operating parameter in a long-term culture.

Diffuse reflectance spectroscopy as a tool for real-time tissue assessment during colorectal cancer surgery

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Baltussen, Elisabeth J. M.; Snaebjornsson, Petur; de Koning, Susan G. Brouwer; Sterenborg, Henricus J. C. M.; Aalbers, Arend G. J.; Kok, Niels; Beets, Geerard L.; Hendriks, Benno H. W.; Kuhlmann, Koert F. D.; Ruers, Theo J. M.

2017-10-01

Colorectal surgery is the standard treatment for patients with colorectal cancer. To overcome two of the main challenges, the circumferential resection margin and postoperative complications, real-time tissue assessment could be of great benefit during surgery. In this ex vivo study, diffuse reflectance spectroscopy (DRS) was used to differentiate tumor tissue from healthy surrounding tissues in patients with colorectal neoplasia. DRS spectra were obtained from tumor tissue, healthy colon, or rectal wall and fat tissue, for every patient. Data were randomly divided into training (80%) and test (20%) sets. After spectral band selection, the spectra were classified using a quadratic classifier and a linear support vector machine. Of the 38 included patients, 36 had colorectal cancer and 2 had an adenoma. When the classifiers were applied to the test set, colorectal cancer could be discriminated from healthy tissue with an overall accuracy of 0.95 (±0.03). This study demonstrates the possibility to separate colorectal cancer from healthy surrounding tissue by applying DRS. High classification accuracies were obtained both in homogeneous and inhomogeneous tissues. This is a fundamental step toward the development of a tool for real-time in vivo tissue assessment during colorectal surgery.

Real-time Visualization of Tissue Dynamics during Embryonic Development and Malignant Transformation

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Yamada, Kenneth

Tissues undergo dramatic changes in organization during embryonic development, as well as during cancer progression and invasion. Recent advances in microscopy now allow us to visualize and track directly the dynamic movements of tissues, their constituent cells, and cellular substructures. This behavior can now be visualized not only in regular tissue culture on flat surfaces (`2D' environments), but also in a variety of 3D environments that may provide physiological cues relevant to understanding dynamics within living organisms. Acquisition of imaging data using various microscopy modalities will provide rich opportunities for determining the roles of physical factors and for computational modeling of complex processes in living tissues. Direct visualization of real-time motility is providing insight into biology spanning multiple spatio-temporal scales. Many cells in our body are known to be in contact with connective tissue and other forms of extracellular matrix. They do so through microscopic cellular adhesions that bind to matrix proteins. In particular, fluorescence microscopy has revealed that cells dynamically probe and bend the matrix at the sites of cell adhesions, and that 3D matrix architecture, stiffness, and elasticity can each regulate migration of the cells. Conversely, cells remodel their local matrix as organs form or tumors invade. Cancer cells can invade tissues using microscopic protrusions that degrade the surrounding matrix; in this case, the local matrix protein concentration is more important for inducing the micro-invasive protrusions than stiffness. On the length scales of tissues, transiently high rates of individual cell movement appear to help establish organ architecture. In fact, isolated cells can self-organize to form tissue structures. In all of these cases, in-depth real-time visualization will ultimately provide the extensive data needed for computer modeling and for testing hypotheses in which physical forces interact

Multivariate reference technique for quantitative analysis of fiber-optic tissue Raman spectroscopy.

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Bergholt, Mads Sylvest; Duraipandian, Shiyamala; Zheng, Wei; Huang, Zhiwei

2013-12-03

We report a novel method making use of multivariate reference signals of fused silica and sapphire Raman signals generated from a ball-lens fiber-optic Raman probe for quantitative analysis of in vivo tissue Raman measurements in real time. Partial least-squares (PLS) regression modeling is applied to extract the characteristic internal reference Raman signals (e.g., shoulder of the prominent fused silica boson peak (~130 cm(-1)); distinct sapphire ball-lens peaks (380, 417, 646, and 751 cm(-1))) from the ball-lens fiber-optic Raman probe for quantitative analysis of fiber-optic Raman spectroscopy. To evaluate the analytical value of this novel multivariate reference technique, a rapid Raman spectroscopy system coupled with a ball-lens fiber-optic Raman probe is used for in vivo oral tissue Raman measurements (n = 25 subjects) under 785 nm laser excitation powers ranging from 5 to 65 mW. An accurate linear relationship (R(2) = 0.981) with a root-mean-square error of cross validation (RMSECV) of 2.5 mW can be obtained for predicting the laser excitation power changes based on a leave-one-subject-out cross-validation, which is superior to the normal univariate reference method (RMSE = 6.2 mW). A root-mean-square error of prediction (RMSEP) of 2.4 mW (R(2) = 0.985) can also be achieved for laser power prediction in real time when we applied the multivariate method independently on the five new subjects (n = 166 spectra). We further apply the multivariate reference technique for quantitative analysis of gelatin tissue phantoms that gives rise to an RMSEP of ~2.0% (R(2) = 0.998) independent of laser excitation power variations. This work demonstrates that multivariate reference technique can be advantageously used to monitor and correct the variations of laser excitation power and fiber coupling efficiency in situ for standardizing the tissue Raman intensity to realize quantitative analysis of tissue Raman measurements in vivo, which is particularly appealing in

Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.).

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You, Yanchun; Xie, Miao; Vasseur, Liette; You, Minsheng

2018-05-01

Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and β-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

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Burgos Lorenzo

2010-07-01

Full Text Available Abstract Background The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently. Results We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (nptII transgene as well as of an internal control (β-actin, used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the nptII transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the gus marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus. Conclusions We have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot

Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer

DEFF Research Database (Denmark)

Reis, Patricia P; Rogatto, Silvia R; Kowalski, Luiz P

2002-01-01

Quantitative real time PCR was performed on genomic DNA from 40 primary oral carcinomas and the normal adjacent tissues. The target genes ECGFB, DIA1, BIK, and PDGFB and the microsatellite markers D22S274 and D22S277, mapped on 22q13, were selected according to our previous loss of heterozygosity...... findings in head and neck tumors. Quantitative PCR relies on the comparison of the amount of product generated from a target gene and that generated from a disomic reference gene (GAPDH-housekeeping gene). Reactions have been performed with normal control in triplicates, using the 7700 Sequence Detection.......0018) for patients with DIA1 gene loss. Relative copy number losses detected in these sequences may be related to disease progression and a worse prognosis in patients with oral cancer....

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

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Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

2017-01-01

A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

Diagnosis of aerobic vaginitis by quantitative real-time PCR.

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Rumyantseva, T A; Bellen, G; Savochkina, Y A; Guschin, A E; Donders, G G G

2016-07-01

To evaluate a real-time PCR-based technique to quantify bacteria associated with aerobic vaginitis (AV) as a potential test. Vaginal samples from 100 women were tested by wet-mount microscopy, gram stain and quantitative real-time PCR targeting Enterobacteriacea, Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli, Streptococcus agalactiae, S. aureus; Lactobacillus spp. AV diagnosis obtained by wet-mount microscopy was used as reference. Some level of AV was diagnosed in 23 (23.7 %) cases. Various concentrations of Enterobacteriacea, Staphylococcus spp., Streptococcus spp. were detected an all patients. Enterococcus spp. were detected in 76 (78.3 %) cases. Summarized concentrations of aerobes were tenfold higher in AV-positive compared to AV-negative cases [7.30lg vs 6.06lg (p = 0.02)]. Concentrations of aerobes in severe, moderate and light AV cases did not vary significantly (p = 0.14). Concentration of lactobacilli was 1000-fold lower in AV-positive cases compared to normal cases (5.3lg vs 8.3lg, p AV-positive cases [19/22 (86.4 %) samples]. The relation of high loads of aerobes to the low numbers of Lactobacilli are a reliable marker for the presence of AV and could substitute microscopy as a test. PCR may be a good standardized substitution for AV diagnosis in settings where well-trained microscopists are lacking.

Complementary techniques: validation of gene expression data by quantitative real time PCR.

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Provenzano, Maurizio; Mocellin, Simone

2007-01-01

Microarray technology can be considered the most powerful tool for screening gene expression profiles of biological samples. After data mining, results need to be validated with highly reliable biotechniques allowing for precise quantitation of transcriptional abundance of identified genes. Quantitative real time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that support its use as a routine bioinstrumentation for gene level measurement. Currently, qrt-PCR is considered by most experts the most appropriate method to confirm or confute microarray-generated data. The knowledge of the biochemical principles underlying qrt-PCR as well as some related technical issues must be beard in mind when using this biotechnology.

GPU-based real-time soft tissue deformation with cutting and haptic feedback.

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Courtecuisse, Hadrien; Jung, Hoeryong; Allard, Jérémie; Duriez, Christian; Lee, Doo Yong; Cotin, Stéphane

2010-12-01

This article describes a series of contributions in the field of real-time simulation of soft tissue biomechanics. These contributions address various requirements for interactive simulation of complex surgical procedures. In particular, this article presents results in the areas of soft tissue deformation, contact modelling, simulation of cutting, and haptic rendering, which are all relevant to a variety of medical interventions. The contributions described in this article share a common underlying model of deformation and rely on GPU implementations to significantly improve computation times. This consistency in the modelling technique and computational approach ensures coherent results as well as efficient, robust and flexible solutions. Copyright © 2010 Elsevier Ltd. All rights reserved.

The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

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Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

2014-01-01

Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

Directory of Open Access Journals (Sweden)

Jing Cai

Full Text Available Quantitative real-time PCR (qPCR is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD, an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA and nonparametric (Kruskal-Wallis tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

Embryonation of Ostertagia ostertagi eggs affects the outcome of real-time quantitative PCR

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Drag, Markus; Höglund, Johan; Nejsum, Peter

prior to detection and quantification by real-time quantitative polymerase chain reaction (qPCR). Fresh O. ostertagi eggs were isolated from cattle faeces and stored at 4°C or 25°C under aerobic or anaerobic conditions. Embryonation was monitored by microscopy and the ITS2 copies were determined by q...... the outcome of qPCR analysis for the quantitative determination of O. ostertagi eggs in cattle faeces. Cold storage at 4°C for up to 3 days or anaerobicvacuum packing at 25°C for up to 336 h will entail no undesirable effects on ITS2 copies....

Embryonation of Ostertagia ostertagi eggs affects the outcome of real-time quantitative PCR

DEFF Research Database (Denmark)

Drag, Markus; Höglund, Johan; Nejsum, Peter

prior to detection and quantification by real-time quantitative polymerase chain reaction (qPCR) . Fresh O. ostertagi eggs were isolated from cattle faeces and stored at 4°C or 25°C under aerobic or anaerobic conditions. Embryonation was monitored by microscopy and the ITS2 copies were determined by q...... the outcome of qPCR analysis for the quantitative determination of O. ostertagi eggs in cattle faeces. Cold storage at 4°C for up to 3 days or anaerobic vacuum packing at 25°C for up to 336 h will entail no undesirable effects on ITS2 copies....

Selection of Suitable Reference Genes for Quantitative Real-time PCR in Sapium sebiferum

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Xue Chen

2017-05-01

Full Text Available Chinese tallow (Sapium sebiferum L. is a promising landscape and bioenergy plant. Measuring gene expression by quantitative real-time polymerase chain reaction (qRT-PCR can provide valuable information on gene function. Stably expressed reference genes for normalization are a prerequisite for ensuring the accuracy of the target gene expression level among different samples. However, the reference genes in Chinese tallow have not been systematically validated. In this study, 12 candidate reference genes (18S, GAPDH, UBQ, RPS15, SAND, TIP41, 60S, ACT7, PDF2, APT, TBP, and TUB were investigated with qRT-PCR in 18 samples, including those from different tissues, from plants treated with sucrose and cold stresses. The data were calculated with four common algorithms, geNorm, BestKeeper, NormFinder, and the delta cycle threshold (ΔCt. TIP41 and GAPDH were the most stable for the tissue-specific experiment, GAPDH and 60S for cold treatment, and GAPDH and UBQ for sucrose stresses, while the least stable genes were 60S, TIP41, and 18S respectively. The comprehensive results showed APT, GAPDH, and UBQ to be the top-ranked stable genes across all the samples. The stability of 60S was the lowest during all experiments. These selected reference genes were further validated by comparing the expression profiles of the chalcone synthase gene in Chinese tallow in different samples. The results will help to improve the accuracy of gene expression studies in Chinese tallow.

qF-SSOP: real-time optical property corrected fluorescence imaging

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Valdes, Pablo A.; Angelo, Joseph P.; Choi, Hak Soo; Gioux, Sylvain

2017-01-01

Fluorescence imaging is well suited to provide image guidance during resections in oncologic and vascular surgery. However, the distorting effects of tissue optical properties on the emitted fluorescence are poorly compensated for on even the most advanced fluorescence image guidance systems, leading to subjective and inaccurate estimates of tissue fluorophore concentrations. Here we present a novel fluorescence imaging technique that performs real-time (i.e., video rate) optical property corrected fluorescence imaging. We perform full field of view simultaneous imaging of tissue optical properties using Single Snapshot of Optical Properties (SSOP) and fluorescence detection. The estimated optical properties are used to correct the emitted fluorescence with a quantitative fluorescence model to provide quantitative fluorescence-Single Snapshot of Optical Properties (qF-SSOP) images with less than 5% error. The technique is rigorous, fast, and quantitative, enabling ease of integration into the surgical workflow with the potential to improve molecular guidance intraoperatively. PMID:28856038

Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.

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Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

2011-01-01

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

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There is a growing interest in the application of human-associated fecal sourceidentification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data q...

Interlaced photoacoustic and ultrasound imaging system with real-time coregistration for ovarian tissue characterization

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Alqasemi, Umar; Li, Hai; Yuan, Guangqian; Kumavor, Patrick; Zanganeh, Saeid; Zhu, Quing

2014-07-01

Coregistered ultrasound (US) and photoacoustic imaging are emerging techniques for mapping the echogenic anatomical structure of tissue and its corresponding optical absorption. We report a 128-channel imaging system with real-time coregistration of the two modalities, which provides up to 15 coregistered frames per second limited by the laser pulse repetition rate. In addition, the system integrates a compact transvaginal imaging probe with a custom-designed fiber optic assembly for in vivo detection and characterization of human ovarian tissue. We present the coregistered US and photoacoustic imaging system structure, the optimal design of the PC interfacing software, and the reconfigurable field programmable gate array operation and optimization. Phantom experiments of system lateral resolution and axial sensitivity evaluation, examples of the real-time scanning of a tumor-bearing mouse, and ex vivo human ovaries studies are demonstrated.

Optimization and real-time control for laser treatment of heterogeneous soft tissues.

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Feng, Yusheng; Fuentes, David; Hawkins, Andrea; Bass, Jon M; Rylander, Marissa Nichole

2009-01-01

Predicting the outcome of thermotherapies in cancer treatment requires an accurate characterization of the bioheat transfer processes in soft tissues. Due to the biological and structural complexity of tumor (soft tissue) composition and vasculature, it is often very difficult to obtain reliable tissue properties that is one of the key factors for the accurate treatment outcome prediction. Efficient algorithms employing in vivo thermal measurements to determine heterogeneous thermal tissues properties in conjunction with a detailed sensitivity analysis can produce essential information for model development and optimal control. The goals of this paper are to present a general formulation of the bioheat transfer equation for heterogeneous soft tissues, review models and algorithms developed for cell damage, heat shock proteins, and soft tissues with nanoparticle inclusion, and demonstrate an overall computational strategy for developing a laser treatment framework with the ability to perform real-time robust calibrations and optimal control. This computational strategy can be applied to other thermotherapies using the heat source such as radio frequency or high intensity focused ultrasound.

Quantitative analysis of the dystrophin gene by real-time PCR

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Maksimovic Nela

2012-01-01

Full Text Available Duchenne and Becker muscular dystrophy (DMD/BMD are severe X-linked neuromuscular disorders caused by mutations in the dystrophin gene. Our aim was to optimize a quantitative real-time PCR method based on SYBR® Green I chemistry for routine diagnostics of DMD/BMD deletion carriers. Twenty female relatives of DMD/BMD patients with previously detected partial gene deletions were studied. The relative quantity of the target exons was calculated by a comparative threshold cycle method (ΔΔCt. The carrier status of all subjects was successfully determined. The gene dosage ratio for non-carriers was 1.07±0.20, and for carriers 0.56±0.11. This assay proved to be simple, rapid, reliable and cost-effective.

Feasibility of iFlow color-coding technique in quantitative real-time measurement of hemodynamic changes after transarterial chemoembolization for hepatocellular carcinoma

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WANG Yuzhe

2018-01-01

Full Text Available Objective To investigate the value of iFlow color-coding technique in quantitative real-time analysis of hemodynamic changes after transarterial chemoembolization (TACE for hepatocellular carcinoma (HCC. Methods A total of 31 patients who were diagnosed with HCC in Shanghai Fifth People′s Hospital from December 2015 to January 2017 were enrolled. No patient underwent surgical operation or ablation. All patients underwent TACE with the same contrast agent, high-pressure injector parameters, and place of angiographic catheter. The iFlow technique was used to generate two-dimensional color-coded images and time-density curve (TDC before and after surgery and measure the opening of the angiographic catheter and the time to peak (TTP of the starting and ending points of the major tumor feeding arteries, as well as the ratio of the areas under the curve (AUC of TDC of tumor tissue and the opening of the angiographic catheter. The paired t-test was used for comparison of continuous data between groups. Results TTP of the major tumor feeding arteries was 4.64±0.49 s before TACE and 5.97±0.84 s after TACE (t=11.57, P＜0.01, and there was a significant difference in AUC between the tumor tissue and the opening of the angiographic catheter (0.53±0.15 vs 0.16±0.12, t=25.85, P＜0.01. There was no significant difference in TTP between the opening of the angiographic catheter and the major tumor feeding arteries before and after TACE (P＞0.05. Before TACE, the TDC of tumor feeding arteries had a shape of “rapid increase-rapid reduction” with relatively high slope and peak value, while after TACE, the TDC had a shape of “increase-flat-reduction” with reductions in slope and peak value. Conclusion The iFlow technique can perform real-time measurement of TTP and TDC of the region of interest and helps with quantitative evaluation of hemodynamic changes in HCC. Therefore, it can provide objective quantitative indices for evaluating the degree of

Real-time optical monitoring of permanent lesion progression in radiofrequency ablated cardiac tissue (Conference Presentation)

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Singh-Moon, Rajinder P.; Hendon, Christine P.

2016-02-01

Despite considerable advances in guidance of radiofrequency ablation (RFA) therapies for atrial fibrillation, success rates have been hampered by an inability to intraoperatively characterize the extent of permanent injury. Insufficient lesions can elusively create transient conduction blockages that eventually reconduct. Prior studies suggest significantly greater met-myoglobin (Mmb) concentrations in the lesion core than those in the healthy myocardium and may serve as a marker for irreversible tissue damage. In this work, we present real-time monitoring of permanent injury through spectroscopic assessment of Mmb concentrations at the catheter tip. Atrial wedges (n=6) were excised from four fresh swine hearts and submerged under pulsatile flow of warm (37oC) phosphate buffered saline. A commercial RFA catheter inserted into a fiber optic sheath allowed for simultaneous measurement of tissue diffuse reflectance (DR) spectra (500-650nm) during application of RF energy. Optical measurements were continuously acquired before, during, and post-ablation, in addition to healthy neighboring tissue. Met-myoglobin, oxy-myoglobin, and deoxy-myoglobin concentrations were extracted from each spectrum using an inverse Monte Carlo method. Tissue injury was validated with Masson's trichrome and hematoxylin and eosin staining. Time courses revealed a rapid increase in tissue Mmb concentrations at the onset of RFA treatment and a gradual plateauing thereafter. Extracted Mmb concentrations were significantly greater post-ablation (p<0.0001) as compared to healthy tissue and correlated well with histological assessment of severe thermal tissue destruction. On going studies are aimed at integrating these findings with prior work on near infrared spectroscopic lesion depth assessment. These results support the use of spectroscopy-facilitated guidance of RFA therapies for real-time permanent injury estimation.

Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR.

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Yogita Maheshwari

Full Text Available Droplet digital polymerase chain reaction (ddPCR is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative PCR (qPCR for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.

Validation of tumor markers in central nervous system germ cell tumors by real-time reverse transcriptase polymerase chain reaction using formalin-fixed paraffin-embedded tissues.

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Kim, Dowhan; Lee, Da Hye; Choi, Junjeong; Shim, Kyu Won; Kim, Se Hoon

2013-01-01

The therapeutic protocols for treatment of germinomas and non-germinomatous germ cell tumors (NGGCTs) are completely different, so it is important to distinguish pure germinomas from NGGCTs. As it can be difficult to diagnose by morphology alone, immunohisto-chemistry (IHC) has been widely used as an ancillary test to improve diagnostic accuracy. However, IHC has limitations due to the misinterpretation of results or the aberrant loss of immunoreactivity. However, real-time RT-PCR has certain advantages over IHC, including its quantitative nature. The aim of our study was to evaluate the usefulness of real-time RT-PCR on formalin-fixed paraffin-embedded (FFPE) tissue blocks for the diagnosis of germ cell tumors of the central nervous system. We selected eight markers of germ cell tumors using a literature search, and validated them using real-time RT-PCR. Among them, POU5F1, NANOG and TGFB2 were statistically significant (P=0.05) in multiple comparisons (MANOVA) of three groups (pure germinomas, mature teratomas and malignant germ cell tumors). Two-group (pure germinomas and NGGCTs) discriminant analysis achieved a 70.0% success rate in cross-validation. We concluded that real-time RT-PCR using FFPE tissue has adequate validating power comparable to IHC in the diagnosis of central nervous system germ cell tumors; therefore, when IHC is not available, not conclusive or not informative, RT-PCR is a potential alternative to a repeat biopsy.

Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species

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Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

2016-01-01

Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management. PMID:27562455

[Research progress on real-time deformable models of soft tissues for surgery simulation].

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Xu, Shaoping; Liu, Xiaoping; Zhang, Hua; Luo, Jie

2010-04-01

Biological tissues generally exhibit nonlinearity, anisotropy, quasi-incompressibility and viscoelasticity about material properties. Simulating the behaviour of elastic objects in real time is one of the current objectives of virtual surgery simulation which is still a challenge for researchers to accurately depict the behaviour of human tissues. In this paper, we present a classification of the different deformable models that have been developed. We present the advantages and disadvantages of each one. Finally, we make a comparison of deformable models and perform an evaluation of the state of the art and the future of deformable models.

Real-time high-resolution measurement of collagen alignment in dynamically loaded soft tissue.

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York, Timothy; Kahan, Lindsey; Lake, Spencer P; Gruev, Viktor

2014-06-01

A technique for creating maps of the direction and strength of fiber alignment in collagenous soft tissues is presented. The method uses a division of focal plane polarimeter to measure circularly polarized light transmitted through the tissue. The architecture of the sensor allows measurement of the retardance and fiber alignment at the full frame rate of the sensor without any moving optics. The technique compares favorably to the standard method of using a rotating polarizer. How the new technique enables real-time capture of the full angular spread of fiber alignment and retardance under various cyclic loading conditions is illustrated.

Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR.

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Furet, Jean-Pierre; Quénée, Pascal; Tailliez, Patrick

2004-12-15

Real-time quantitative PCR assays were developed for the absolute quantification of lactic acid bacteria (LAB) (Streptococcus thermophilus, Lactobacillus delbrueckii, L. casei, L. paracasei, L. rhamnosus, L. acidophilus and L. johnsonii) in fermented milk products. The results of molecular quantification and classic bacterial enumeration did not differ significantly with respect to S. thermophilus and the species of the L. casei group which were detected in the six commercial fermented products tested, thus showing that DNA extraction was efficient and that genomic DNA solutions were free of PCR inhibitors. For L. delbrueckii, the results of bacterial enumeration were generally lower by a factor 10 to 100 than those of PCR quantification, suggesting a loss of viability during storage of the dairy products at 1-8 degrees C for most of the strains in this species. Real-time quantitative assays enabled identification of the species of lactic acid bacterial strains initially present in commercial fermented milk products and their accurate quantification with a detection threshold of 10(3) cells per ml of product.

Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos

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Van Zeveren Alex

2005-12-01

Full Text Available Abstract Background Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used. Results In this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages. Conclusion Using the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

Quantitative detection of Campylobacter jejuni on fresh chicken carcasses by real-time PCR.

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Rönner, Anna-Clara; Lindmark, Hans

2007-06-01

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 microl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

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Takabatake, Reona; Koiwa, Tomohiro; Kasahara, Masaki; Takashima, Kaori; Futo, Satoshi; Minegishi, Yasutaka; Akiyama, Hiroshi; Teshima, Reiko; Oguchi, Taichi; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

2011-01-01

To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

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Qiu, Reng; Sun, Boguang; Fang, Shasha; Sun, Li; Liu, Xiao

2013-03-01

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

Quantitative assessment of hematopoietic chimerism by quantitative real-time polymerase chain reaction of sequence polymorphism systems after hematopoietic stem cell transplantation.

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Qin, Xiao-ying; Li, Guo-xuan; Qin, Ya-zhen; Wang, Yu; Wang, Feng-rong; Liu, Dai-hong; Xu, Lan-ping; Chen, Huan; Han, Wei; Wang, Jing-zhi; Zhang, Xiao-hui; Li, Jin-lan; Li, Ling-di; Liu, Kai-yan; Huang, Xiao-jun

2011-08-01

Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT. A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally. Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method. This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.

Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

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Zhang, Jin-Ju; Xu, Jing-Feng; Shen, Yong-Wei; Ma, Shi-Jiao; Zhang, Ting-Ting; Meng, Qing-Lin; Lan, Wen-Jun; Zhang, Chun; Liu, Xiao-Mei

2017-07-01

Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Real-time deformation of human soft tissues: A radial basis meshless 3D model based on Marquardt's algorithm.

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Zhou, Jianyong; Luo, Zu; Li, Chunquan; Deng, Mi

2018-01-01

When the meshless method is used to establish the mathematical-mechanical model of human soft tissues, it is necessary to define the space occupied by human tissues as the problem domain and the boundary of the domain as the surface of those tissues. Nodes should be distributed in both the problem domain and on the boundaries. Under external force, the displacement of the node is computed by the meshless method to represent the deformation of biological soft tissues. However, computation by the meshless method consumes too much time, which will affect the simulation of real-time deformation of human tissues in virtual surgery. In this article, the Marquardt's Algorithm is proposed to fit the nodal displacement at the problem domain's boundary and obtain the relationship between surface deformation and force. When different external forces are applied, the deformation of soft tissues can be quickly obtained based on this relationship. The analysis and discussion show that the improved model equations with Marquardt's Algorithm not only can simulate the deformation in real-time but also preserve the authenticity of the deformation model's physical properties. Copyright © 2017 Elsevier B.V. All rights reserved.

Decay Of Bacterial Pathogens, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure-Amended Soils

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This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manure-amended agricultural soils. Known concentrations of transformed green...

Decay Of Bacterial Pathogen, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure Amended Soils

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This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria, and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manre-amended agricultural soils. Known concentrations of transformed green fluore...

EVALUATION OF A RAPID, QUANTITATIVE REAL-TIME PCR METHOD FOR ENUMERATION OF PATHOGENIC CANDIDA CELLS IN WATER

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Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan?) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C....

Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA.

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Majid, Farjana; Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

2014-01-01

Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log 10 copies/ml and 6.95 ± 1.08 log 10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log 10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35.

Use of quantitative real-time PCR for direct detection of serratia marcescens in marine and other aquatic environments.

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Joyner, Jessica; Wanless, David; Sinigalliano, Christopher D; Lipp, Erin K

2014-03-01

Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml(-1) and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml(-1). This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.

Quantified Facial Soft-tissue Strain in Animation Measured by Real-time Dynamic 3-Dimensional Imaging.

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Hsu, Vivian M; Wes, Ari M; Tahiri, Youssef; Cornman-Homonoff, Joshua; Percec, Ivona

2014-09-01

The aim of this study is to evaluate and quantify dynamic soft-tissue strain in the human face using real-time 3-dimensional imaging technology. Thirteen subjects (8 women, 5 men) between the ages of 18 and 70 were imaged using a dual-camera system and 3-dimensional optical analysis (ARAMIS, Trilion Quality Systems, Pa.). Each subject was imaged at rest and with the following facial expressions: (1) smile, (2) laughter, (3) surprise, (4) anger, (5) grimace, and (6) pursed lips. The facial strains defining stretch and compression were computed for each subject and compared. The areas of greatest strain were localized to the midface and lower face for all expressions. Subjects over the age of 40 had a statistically significant increase in stretch in the perioral region while lip pursing compared with subjects under the age of 40 (58.4% vs 33.8%, P = 0.015). When specific components of lip pursing were analyzed, there was a significantly greater degree of stretch in the nasolabial fold region in subjects over 40 compared with those under 40 (61.6% vs 32.9%, P = 0.007). Furthermore, we observed a greater degree of asymmetry of strain in the nasolabial fold region in the older age group (18.4% vs 5.4%, P = 0.03). This pilot study illustrates that the face can be objectively and quantitatively evaluated using dynamic major strain analysis. The technology of 3-dimensional optical imaging can be used to advance our understanding of facial soft-tissue dynamics and the effects of animation on facial strain over time.

Real-time in vivo tissue characterization with diffuse reflectance spectroscopy during transthoracic lung biopsy: a clinical feasibility study

NARCIS (Netherlands)

Spliethoff, Jarich; Prevoo, Warner; Meier, Mark A.J.; de Jong, Jeroen; Evers, Daniel; Evers, Daniel J.; Sterenborg, Hendricus J.C.M.; Lucassen, Gerald; Lucassen, Gerald W.; Hendriks, Benno H.W.; Ruers, Theo J.M.

2016-01-01

Purpose: This study presents the first in vivo real-time tissue characterization during image-guided percutaneous lung biopsies using diffuse reflectance spectroscopy (DRS) sensing at the tip of a biopsy needle with integrated optical fibers. Experimental Design: Tissues from 21 consented patients

Quantified Facial Soft-tissue Strain in Animation Measured by Real-time Dynamic 3-Dimensional Imaging

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Vivian M. Hsu, MD

2014-09-01

Conclusions: This pilot study illustrates that the face can be objectively and quantitatively evaluated using dynamic major strain analysis. The technology of 3-dimensional optical imaging can be used to advance our understanding of facial soft-tissue dynamics and the effects of animation on facial strain over time.

Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae)

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Fu, Wei; Xie, Wen; Zhang, Zhuo; Wang, Shaoli; Wu, Qingjun; Liu, Yong; Zhou, Xiaomao; Zhou, Xuguo; Zhang, Youjun

2013-01-01

Abstract: Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest. PMID:23983612

Real-time surgical simulation for deformable soft-tissue objects with a tumour using Boundary Element techniques

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Wang, P.; Becker, A. A.; Jones, I. A.; Glover, A. T.; Benford, S. D.; Vloeberghs, M.

2009-08-01

A virtual-reality real-time simulation of surgical operations that incorporates the inclusion of a hard tumour is presented. The software is based on Boundary Element (BE) technique. A review of the BE formulation for real-time analysis of two-domain deformable objects, using the pre-solution technique, is presented. The two-domain BE software is incorporated into a surgical simulation system called VIRS to simulate the initiation of a cut on the surface of the soft tissue and extending the cut deeper until the tumour is reached.

Real-time surgical simulation for deformable soft-tissue objects with a tumour using Boundary Element techniques

International Nuclear Information System (INIS)

Wang, P; Becker, A A; Jones, I A; Glover, A T; Benford, S D; Vloeberghs, M

2009-01-01

A virtual-reality real-time simulation of surgical operations that incorporates the inclusion of a hard tumour is presented. The software is based on Boundary Element (BE) technique. A review of the BE formulation for real-time analysis of two-domain deformable objects, using the pre-solution technique, is presented. The two-domain BE software is incorporated into a surgical simulation system called VIRS to simulate the initiation of a cut on the surface of the soft tissue and extending the cut deeper until the tumour is reached.

Real-Time Electrical Bioimpedance Characterization of Neointimal Tissue for Stent Applications

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David Rivas-Marchena

2017-07-01

Full Text Available To follow up the restenosis in arteries stented during an angioplasty is an important current clinical problem. A new approach to monitor the growth of neointimal tissue inside the stent is proposed on the basis of electrical impedance spectroscopy (EIS sensors and the oscillation-based test (OBT circuit technique. A mathematical model was developed to analytically describe the histological composition of the neointima, employing its conductivity and permittivity data. The bioimpedance model was validated against a finite element analysis (FEA using COMSOL Multiphysics software. A satisfactory correlation between the analytical model and FEA simulation was achieved in most cases, detecting some deviations introduced by the thin “double layer” that separates the neointima and the blood. It is hereby shown how to apply conformal transformations to obtain bioimpedance electrical models for stack-layered tissues over coplanar electrodes. Particularly, this can be applied to characterize the neointima in real-time. This technique is either suitable as a main mechanism for restenosis follow-up or it can be combined with proposed intelligent stents for blood pressure measurements to auto-calibrate the sensibility loss caused by the adherence of the tissue on the micro-electro-mechanical sensors (MEMSs.

Telerobotic system concept for real-time soft-tissue imaging during radiotherapy beam delivery.

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Schlosser, Jeffrey; Salisbury, Kenneth; Hristov, Dimitre

2010-12-01

The curative potential of external beam radiation therapy is critically dependent on having the ability to accurately aim radiation beams at intended targets while avoiding surrounding healthy tissues. However, existing technologies are incapable of real-time, volumetric, soft-tissue imaging during radiation beam delivery, when accurate target tracking is most critical. The authors address this challenge in the development and evaluation of a novel, minimally interfering, telerobotic ultrasound (U.S.) imaging system that can be integrated with existing medical linear accelerators (LINACs) for therapy guidance. A customized human-safe robotic manipulator was designed and built to control the pressure and pitch of an abdominal U.S. transducer while avoiding LINAC gantry collisions. A haptic device was integrated to remotely control the robotic manipulator motion and U.S. image acquisition outside the LINAC room. The ability of the system to continuously maintain high quality prostate images was evaluated in volunteers over extended time periods. Treatment feasibility was assessed by comparing a clinically deployed prostate treatment plan to an alternative plan in which beam directions were restricted to sectors that did not interfere with the transabdominal U.S. transducer. To demonstrate imaging capability concurrent with delivery, robot performance and U.S. target tracking in a phantom were tested with a 15 MV radiation beam active. Remote image acquisition and maintenance of image quality with the haptic interface was successfully demonstrated over 10 min periods in representative treatment setups of volunteers. Furthermore, the robot's ability to maintain a constant probe force and desired pitch angle was unaffected by the LINAC beam. For a representative prostate patient, the dose-volume histogram (DVH) for a plan with restricted sectors remained virtually identical to the DVH of a clinically deployed plan. With reduced margins, as would be enabled by real-time

Telerobotic system concept for real-time soft-tissue imaging during radiotherapy beam delivery

International Nuclear Information System (INIS)

Schlosser, Jeffrey; Salisbury, Kenneth; Hristov, Dimitre

2010-01-01

Purpose: The curative potential of external beam radiation therapy is critically dependent on having the ability to accurately aim radiation beams at intended targets while avoiding surrounding healthy tissues. However, existing technologies are incapable of real-time, volumetric, soft-tissue imaging during radiation beam delivery, when accurate target tracking is most critical. The authors address this challenge in the development and evaluation of a novel, minimally interfering, telerobotic ultrasound (U.S.) imaging system that can be integrated with existing medical linear accelerators (LINACs) for therapy guidance. Methods: A customized human-safe robotic manipulator was designed and built to control the pressure and pitch of an abdominal U.S. transducer while avoiding LINAC gantry collisions. A haptic device was integrated to remotely control the robotic manipulator motion and U.S. image acquisition outside the LINAC room. The ability of the system to continuously maintain high quality prostate images was evaluated in volunteers over extended time periods. Treatment feasibility was assessed by comparing a clinically deployed prostate treatment plan to an alternative plan in which beam directions were restricted to sectors that did not interfere with the transabdominal U.S. transducer. To demonstrate imaging capability concurrent with delivery, robot performance and U.S. target tracking in a phantom were tested with a 15 MV radiation beam active. Results: Remote image acquisition and maintenance of image quality with the haptic interface was successfully demonstrated over 10 min periods in representative treatment setups of volunteers. Furthermore, the robot's ability to maintain a constant probe force and desired pitch angle was unaffected by the LINAC beam. For a representative prostate patient, the dose-volume histogram (DVH) for a plan with restricted sectors remained virtually identical to the DVH of a clinically deployed plan. With reduced margins, as

Clinical Usefulness of Real-Time Polymerase Chain Reaction for the Diagnosis of Vibrio vulnificus Infection Using Skin and Soft Tissues.

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Lee, Jun-Young; Kim, Seok Won; Kim, Dong-Min; Yun, Na Ra; Kim, Choon-Mee; Lee, Sang-Hong

2017-08-01

Vibrio vulnificus is a halophilic gram-negative bacillus isolated in seawater, fish, and shellfish. Infection by V. vulnificus is the most severe food-borne infection reported in the United States of America. Here, we aimed to examine the clinical usefulness of polymerase chain reaction (PCR) using tissue specimens other than blood samples as a diagnostic tool for V. vulnificus infection. A retrospective study was conducted with patients who underwent real-time PCR of toxR in both blood and skin tissues, including serum, bullae, swab, and operation room specimens, between 2006 and 2009. The median V. vulnificus DNA load of 14 patients in real-time PCR analysis of serum at the time of admission was 638.5 copies/mL blood, which was within the interquartile range (IQR: 37-3,225). In contrast, the median value by real-time PCR using the first tissue specimen at the time of admission was 16,650 copies/mL tissue fluid (IQR: 4,419-832,500). This difference was statistically significant ( P = 0.022). DNA copy numbers in tissues were less affected by short-term antibiotic administration than that in blood samples, and antibiotic administration increased the DNA copy number in some patients. We found, for the first time, that DNA copy numbers in tissues of patients infected by V. vulnificus were higher than those in blood samples. Additionally, skin lesions were more useful than blood samples as specimens for PCR analysis in patients administered antibiotics for V. vulnificus infection before admission.

Rapid quantitative detection of Lactobacillus sakei in meat and fermented sausages by real-time PCR.

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Martín, Belén; Jofré, Anna; Garriga, Margarita; Pla, Maria; Aymerich, Teresa

2006-09-01

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.

Legionellosis and Lung Abscesses: Contribution of Legionella Quantitative Real-Time PCR to an Adapted Followup

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G. Descours

2013-01-01

Full Text Available We report a case of severe Legionnaires' disease (LD complicated by a lung abscess in an immunocompetent patient who required ECMO therapy and thoracic surgery. The results of repeated Legionella quantitative real-time PCR performed on both sera and respiratory samples correlated with the LD severity and the poor clinical outcome. Moreover, the PCR allowed for the detection of Legionella DNA in the lung abscess specimen, which was negative when cultured for Legionella. This case report provides a logical basis for further investigations to examine whether the Legionella quantitative PCR could improve the assessment of LD severity and constitute a prognostic marker.

Application of quantitative real-time PCR compared to filtration methods for the enumeration of Escherichia coli in surface waters within Vietnam.

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Vital, Pierangeli G; Van Ha, Nguyen Thi; Tuyet, Le Thi Hong; Widmer, Kenneth W

2017-02-01

Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

Real-time simulation of biological soft tissues: a PGD approach.

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Niroomandi, S; González, D; Alfaro, I; Bordeu, F; Leygue, A; Cueto, E; Chinesta, F

2013-05-01

We introduce here a novel approach for the numerical simulation of nonlinear, hyperelastic soft tissues at kilohertz feedback rates necessary for haptic rendering. This approach is based upon the use of proper generalized decomposition techniques, a generalization of PODs. Proper generalized decomposition techniques can be considered as a means of a priori model order reduction and provides a physics-based meta-model without the need for prior computer experiments. The suggested strategy is thus composed of an offline phase, in which a general meta-model is computed, and an online evaluation phase in which the results are obtained at real time. Results are provided that show the potential of the proposed technique, together with some benchmark test that shows the accuracy of the method. Copyright © 2013 John Wiley & Sons, Ltd.

Quantitation of hepatitis B virus DNA in plasma using a sensitive cost-effective "in-house" real-time PCR assay

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Daniel Hubert Darius

2009-01-01

Full Text Available Background: Sensitive nucleic acid testing for the detection and accurate quantitation of hepatitis B virus (HBV is necessary to reduce transmission through blood and blood products and for monitoring patients on antiviral therapy. The aim of this study is to standardize an "in-house" real-time HBV polymerase chain reaction (PCR for accurate quantitation and screening of HBV. Materials and Methods: The "in-house" real-time assay was compared with a commercial assay using 30 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, hepatitis C virus (HCV antibody and human immunodeficiency virus (HIV antibody. Further, 30 HBV-genotyped samples were tested to evaluate the "in-house" assay′s capacity to detect genotypes prevalent among individuals attending this tertiary care hospital. Results: The lower limit of detection of this "in-house" HBV real-time PCR was assessed against the WHO international standard and found to be 50 IU/mL. The interassay and intra-assay coefficient of variation (CV of this "in-house" assay ranged from 1.4% to 9.4% and 0.0% to 2.3%, respectively. Virus loads as estimated with this "in-house" HBV real-time assay correlated well with the commercial artus HBV RG PCR assay ( r = 0.95, P < 0.0001. Conclusion: This assay can be used for the detection and accurate quantitation of HBV viral loads in plasma samples. This assay can be employed for the screening of blood donations and can potentially be adapted to a multiplex format for simultaneous detection of HBV, HIV and HCV to reduce the cost of testing in blood banks.

Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

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Tandale Babasaheb V

2008-12-01

Full Text Available Abstract Background Chandipura virus (CHPV, a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR using TaqMan technology was developed for rapid diagnosis. Methods Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice in ovo (eggs, in vitro (Vero E6, PS, RD and Sand fly cell line] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard. Results Real-time one step RT-PCR was optimized using in vitro transcribed (IVT RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99 with maximum Coefficient of variation (CV = 5.91% for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 102 PFU/ml. Vero and PS cell-lines (1.2 × 103 PFU/ml were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy

An effective method for the quantitative detection of porcine endogenous retrovirus in pig tissues.

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Zhang, Peng; Yu, Ping; Wang, Wei; Zhang, Li; Li, Shengfu; Bu, Hong

2010-05-01

Xenotransplantation shows great promise for providing a virtually limitless supply of cells, tissues, and organs for a variety of therapeutical procedures. However, the potential of porcine endogenous retrovirus (PERV) as a human-tropic pathogen, particularly as a public health risk, is a major concern for xenotransplantation. This study focus on the detection of copy number in various tissues and organs in Banna Minipig Inbreed (BMI) from 2006 to 2007 in West China Hospital, Sichuan University. Real-time quantitative polymerase chain reaction (SYBR Green I) was performed in this study. The results showed that the pol gene had the most copy number in tissues compared with gag, envA, and envB. Our experiment will offer a rapid and accurate method for the detection of the copy number in various tissues and was especially suitable for the selection of tissues or organs in future clinical xenotransplantation.

A human fecal contamination index for ranking impaired recreational watersusing the HF183 quantitative real-time PCR method

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Human fecal pollution of surface water remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for recreational water quality risk managem...

Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer

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Miller Nicola

2007-11-01

Full Text Available Abstract Background Real-time quantitative PCR (RQ-PCR forms the basis of many breast cancer biomarker studies and novel prognostic assays, paving the way towards personalised cancer treatments. Normalisation of relative RQ-PCR data is required to control for non-biological variation introduced during sample preparation. Endogenous control (EC genes, used in this context, should ideally be expressed constitutively and uniformly across treatments in all test samples. Despite widespread recognition that the accuracy of the normalised data is largely dependent on the reliability of the EC, there are no reports of the systematic validation of genes commonly used for this purpose in the analysis of gene expression by RQ-PCR in primary breast cancer tissues. The aim of this study was to identify the most suitable endogenous control genes for RQ-PCR analysis of primary breast tissue from a panel of eleven candidates in current use. Oestrogen receptor alpha (ESR1 was used a target gene to compare the effect of choice of EC on the estimate of gene quantity. Results The expression and validity of candidate ECs (GAPDH, TFRC, ABL, PPIA, HPRT1, RPLP0, B2M, GUSB, MRPL19, PUM1 and PSMC4 was determined in 6 benign and 21 malignant primary breast cancer tissues. Gene expression data was analysed using two different statistical models. MRPL19 and PPIA were identified as the most stable and reliable EC genes, while GUSB, RPLP0 and ABL were least stable. There was a highly significant difference in variance between ECs. ESR1 expression was appreciably higher in malignant compared to benign tissues and there was a significant effect of EC on the magnitude of the error associated with the relative quantity of ESR1. Conclusion We have validated two endogenous control genes, MRPL19 and PPIA, for RQ-PCR analysis of gene expression in primary breast tissue. Of the genes in current use in this field, the above combination offers increased accuracy and resolution in the

Quantitative real-time monitoring of multi-elements in airborne particulates by direct introduction into an inductively coupled plasma mass spectrometer

International Nuclear Information System (INIS)

Suzuki, Yoshinari; Sato, Hikaru; Hiyoshi, Katsuhiro; Furuta, Naoki

2012-01-01

A new calibration system for real-time determination of trace elements in airborne particulates was developed. Airborne particulates were directly introduced into an inductively coupled plasma mass spectrometer, and the concentrations of 15 trace elements were determined by means of an external calibration method. External standard solutions were nebulized by an ultrasonic nebulizer (USN) coupled with a desolvation system, and the resulting aerosol was introduced into the plasma. The efficiency of sample introduction via the USN was calculated by two methods: (1) the introduction of a Cr standard solution via the USN was compared with introduction of a Cr(CO) 6 standard gas via a standard gas generator and (2) the aerosol generated by the USN was trapped on filters and then analyzed. The Cr introduction efficiencies obtained by the two methods were the same, and the introduction efficiencies of the other elements were equal to the introduction efficiency of Cr. Our results indicated that our calibration method for introduction efficiency worked well for the 15 elements (Ti, V, Cr, Mn, Co, Ni, Cu, Zn, As, Mo, Sn, Sb, Ba, Tl and Pb). The real-time data and the filter-collection data agreed well for elements with low-melting oxides (V, Co, As, Mo, Sb, Tl, and Pb). In contrast, the real-time data were smaller than the filter-collection data for elements with high-melting oxides (Ti, Cr, Mn, Ni, Cu, Zn, Sn, and Ba). This result implies that the oxides of these 8 elements were not completely fused, vaporized, atomized, and ionized in the initial radiation zone of the inductively coupled plasma. However, quantitative real-time monitoring can be realized after correction for the element recoveries which can be calculated from the ratio of real-time data/filter-collection data. - Highlights: ► APs were directly introduced into ICP-MS and real-time analysis was performed. ► The real-time data were calibrated by a multi-element standard solution from USN. ► During real-time

Applying a Real-Time PCR Assay for Histoplasma capsulatum to Clinically Relevant Formalin-Fixed Paraffin-Embedded Human Tissue

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Koepsell, Scott A.; Hinrichs, Steven H.

2012-01-01

A real-time PCR assay to detect Histoplasma capsulatum in formalin-fixed, paraffin-embedded (FFPE) tissue is described. The assay had an analytical sensitivity of 6 pg/μl of fungal DNA, analytical specificity of 100%, and clinical sensitivity of 88.9%. This proof-of-concept study may aid in the diagnosis of histoplasmosis from FFPE tissue. PMID:22855519

Comparative study of standard space and real space analysis of quantitative MR brain data.

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Aribisala, Benjamin S; He, Jiabao; Blamire, Andrew M

2011-06-01

To compare the robustness of region of interest (ROI) analysis of magnetic resonance imaging (MRI) brain data in real space with analysis in standard space and to test the hypothesis that standard space image analysis introduces more partial volume effect errors compared to analysis of the same dataset in real space. Twenty healthy adults with no history or evidence of neurological diseases were recruited; high-resolution T(1)-weighted, quantitative T(1), and B(0) field-map measurements were collected. Algorithms were implemented to perform analysis in real and standard space and used to apply a simple standard ROI template to quantitative T(1) datasets. Regional relaxation values and histograms for both gray and white matter tissues classes were then extracted and compared. Regional mean T(1) values for both gray and white matter were significantly lower using real space compared to standard space analysis. Additionally, regional T(1) histograms were more compact in real space, with smaller right-sided tails indicating lower partial volume errors compared to standard space analysis. Standard space analysis of quantitative MRI brain data introduces more partial volume effect errors biasing the analysis of quantitative data compared to analysis of the same dataset in real space. Copyright © 2011 Wiley-Liss, Inc.

Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

Science.gov (United States)

Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

2013-02-01

The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

Development Status of the WetLab-2 Project: New Tools for On-orbit Real-time Quantitative Gene Expression.

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Jung, Jimmy; Parra, Macarena P.; Almeida, Eduardo; Boone, Travis; Chinn, Tori; Ricco, Antonio; Souza, Kenneth; Hyde, Liz; Rukhsana, Yousuf; Richey, C. Scott

2013-01-01

The primary objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform to facilitate gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens grown or cultured on orbit. The WetLab-2 equipment will be capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. In addition to the logistical benefits of in-situ sample processing and analysis, conducting qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms. The system can also validate terrestrial analyses of samples returned from ISS by providing quantitative on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experimental parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Finally, WetLab-2 can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-5 in Aug. 2014.Progress to date: The WetLab-2 project completed a thorough study of commercially available qRT-PCR systems and performed a downselect based on both scientific and engineering requirements. The selected instrument, the Cepheid SmartCycler, has advantages including modular design (16 independent PCR modules), low power consumption, and rapid ramp times. The SmartCycler has multiplex capabilities, assaying up to four genes of interest in each of the 16 modules. The WetLab-2 team is currently working with Cepheid to modify the unit for housing within an EXPRESS rack locker on the ISS. This will enable the downlink of data to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project is

Accounting for large deformations in real-time simulations of soft tissues based on reduced-order models.

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Niroomandi, S; Alfaro, I; Cueto, E; Chinesta, F

2012-01-01

Model reduction techniques have shown to constitute a valuable tool for real-time simulation in surgical environments and other fields. However, some limitations, imposed by real-time constraints, have not yet been overcome. One of such limitations is the severe limitation in time (established in 500Hz of frequency for the resolution) that precludes the employ of Newton-like schemes for solving non-linear models as the ones usually employed for modeling biological tissues. In this work we present a technique able to deal with geometrically non-linear models, based on the employ of model reduction techniques, together with an efficient non-linear solver. Examples of the performance of the technique over some examples will be given. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Development of a non invasion real-time PCR assay for the quantitation of chicken parvovirus in fecal swabs

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The present study describes the development of a real time Taqman polymerase chain reaction (PCR) assay using a fluorescent labeled probe for the detection and quantitation of chicken parvovirus (ChPV) in feces. The primers and probes were designed based on the nucleotide sequence of the non struct...

Finite Element Methods for real-time Haptic Feedback of Soft-Tissue Models in Virtual Reality Simulators

Science.gov (United States)

Frank, Andreas O.; Twombly, I. Alexander; Barth, Timothy J.; Smith, Jeffrey D.; Dalton, Bonnie P. (Technical Monitor)

2001-01-01

We have applied the linear elastic finite element method to compute haptic force feedback and domain deformations of soft tissue models for use in virtual reality simulators. Our results show that, for virtual object models of high-resolution 3D data (>10,000 nodes), haptic real time computations (>500 Hz) are not currently possible using traditional methods. Current research efforts are focused in the following areas: 1) efficient implementation of fully adaptive multi-resolution methods and 2) multi-resolution methods with specialized basis functions to capture the singularity at the haptic interface (point loading). To achieve real time computations, we propose parallel processing of a Jacobi preconditioned conjugate gradient method applied to a reduced system of equations resulting from surface domain decomposition. This can effectively be achieved using reconfigurable computing systems such as field programmable gate arrays (FPGA), thereby providing a flexible solution that allows for new FPGA implementations as improved algorithms become available. The resulting soft tissue simulation system would meet NASA Virtual Glovebox requirements and, at the same time, provide a generalized simulation engine for any immersive environment application, such as biomedical/surgical procedures or interactive scientific applications.

Comparative evaluation of a laboratory developed real-time PCR assay and the RealStar® HHV-6 PCR Kit for quantitative detection of human herpesvirus 6.

Science.gov (United States)

Yip, Cyril C Y; Sridhar, Siddharth; Cheng, Andrew K W; Fung, Ami M Y; Cheng, Vincent C C; Chan, Kwok-Hung; Yuen, Kwok-Yung

2017-08-01

HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar ® HHV-6 PCR Kit. The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar ® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log 10 copies/ml and a coefficient of determination (R 2 ) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log 10 and 2 log 10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar ® HHV-6 PCR Kit (R 2 =0.926; PPCR Kit. Copyright © 2017 Elsevier B.V. All rights reserved.

A new method to study changes in microvascular blood volume in muscle and adipose tissue: Real time imaging in humans and rat

DEFF Research Database (Denmark)

Sjøberg, Kim Anker; Rattigan, Stephen; Hiscock, Natalie J

2011-01-01

We employed and evaluated a new application of contrast enhanced ultrasound for real time imaging of changes in microvascular blood volume (MVB) in tissues in females, males and rat. Continuous real time imaging was performed using contrast enhanced ultrasound to quantify infused gas filled micro...

Exploring valid reference genes for quantitative real - time rt - pce studies of hydrogenperoxide signaling in arabidopsis

International Nuclear Information System (INIS)

Zhou, H.; Han, B.; Xie, Y.; Zhang, J.; Shen, W.

2015-01-01

Hydrogen peroxide (H/sub 2/O/sub 2/ ) acts as a signaling molecule modulating the expression of various genes in plants. However, the reference gene(s) used for gene expression analysis of H/sub 2/O/sub 2/ signaling is still arbitrary. A reliable result obtained by quantitative real-time RT-PCR (RT-qPCR) highly depends on accurate transcript normalization using stably expressed reference genes, whereas the inaccurate normalization could easily lead to the false conclusions. In this report, by using geNorm and NormFinder algorithms, 12 candidate reference genes were evaluated and compared in root and shoot tissues of Arabidopsis upon different doses of H/sub 2/O/sub 2/. The results revealed that, in our experimental conditions, three novel reference genes (TIP41-like, UKN, and UBC21) were identified and validated as suitable reference genes for RT-qPCR normalization in both root and shoot tissues under oxidative stress. This conclusion was further confirmed by publicly available microarray data of methyl viologen and drought stress. In comparison with a single reference gene (EF-1a), the expression pattern of ZAT12 modulated by H/sub 2/O/sub 2/, when using TIP41-like, UKN, and UBC21 as multiple reference gene(s), was similar with the previous reports by using northern blotting. Thus, we proposed that these three reference genes might be good candidates for other researchers to include in their reference gene validation in gene expression studies under H/sub 2/O/sub 2/ related oxidative stress. (author)

TaqMan MGB probe fluorescence real-time quantitative PCR for rapid detection of Chinese Sacbrood virus.

Directory of Open Access Journals (Sweden)

Ma Mingxiao

Full Text Available Sacbrood virus (SBV is a picorna-like virus that affects honey bees (Apis mellifera and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.

Exploring Valid Reference Genes for Quantitative Real-Time PCR Analysis in Sesamia inferens (Lepidoptera: Noctuidae)

OpenAIRE

Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

2015-01-01

The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study...

[Quantitative fluorogenic real-time PCR assay for respiratory syncytial virus detection].

Science.gov (United States)

Zhang, Qi-wei; You, Shang-you; Sun, Ji-min; Wu, Qi; Yu, Chun-hua; Zhang, Chu-yu

2005-07-01

To Establish a rapid and objective quantitative fluorogenic real-time PCR assay for early detection of human respiratory syncytial virus (hRSV). Two pairs of primers and one TaqMan Fluorogenic probe that are specific for the recognition of the most conservative N gene of hRSV for virus detection with LighCycler PCR in 93 nasopharyngeal secretion specimens collected from infants and young children. The assay was compared with virus isolation, routine PCR, nested PCR, and enzyme-linked immunosorbent assay (ELISA). This TaqMan assay had a sensitivity of 1 x 10(2) cDNA copies/microl with a dynamic range between 1 x 10(2) and 1 x 10(7) cDNA copies/microl, which was the same as that of nested PCR, but 10 times more sensitive than routine PCR. The specificity of the assay was evaluated by comparing hRSV with polivirus type 1, coxsackie virus type 2, influenza A, influenza B and adenovirus type 7. A PCR product of the expected size (195 bp) was produced and fluorescence signal detected for hRSV, but not for any of the other viruses. The results in LightCycler and Rotor-Gene instrument were consistent. Forty-four specimens (43.9%) were hRSV-positive with this assay and 4 (4/93,4.3%) were hRSV-positive with ELISA, showing rather low correlation between the two methods. No visible relation was found between the concentration of hRSV RNA and severity of the disease. This assay is rapid, sensitive, specific and quantitative, and has the potential of wide application for early diagnosis of hRSV infection and evaluation of the therapeutic effect.

Fiber optic based multiparametric spectroscopy in vivo: Toward a new quantitative tissue vitality index

Science.gov (United States)

Kutai-Asis, Hofit; Barbiro-Michaely, Efrat; Deutsch, Assaf; Mayevsky, Avraham

2006-02-01

In our previous publication (Mayevsky et al SPIE 5326: 98-105, 2004) we described a multiparametric fiber optic system enabling the evaluation of 4 physiological parameters as indicators of tissue vitality. Since the correlation between the various parameters may differ in various pathophysiological conditions there is a need for an objective quantitative index that will integrate the relative changes measured in real time by the multiparametric monitoring system into a single number-vitality index. Such an approach to calculate tissue vitality index is critical for the possibility to use such an instrument in clinical environments. In the current presentation we are reporting our preliminary results indicating that calculation of an objective tissue vitality index is feasible. We used an intuitive empirical approach based on the comparison between the calculated index by the computer and the subjective evaluation made by an expert in the field of physiological monitoring. We used the in vivo brain of rats as an animal model in our current studies. The rats were exposed to anoxia, ischemia and cortical spreading depression and the responses were recorded in real time. At the end of the monitoring session the results were analyzed and the tissue vitality index was calculated offline. Mitochondrial NADH, tissue blood flow and oxy-hemoglobin were used to calculate the vitality index of the brain in vivo, where each parameter received a different weight, in each experiment type based on their significance. It was found that the mitochondrial NADH response was the main factor affected the calculated vitality index.

Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus

Directory of Open Access Journals (Sweden)

Kai Wang

2017-04-01

Full Text Available Quantitative real-time reverse transcription PCR (RT-qPCR has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes in Amorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genes Amorphophallus were cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging in A. albus and A. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated that EF1-a, EIF4A, H3 and UBQ were the best reference genes under heat stress in Amorphophallus. Furthermore, EF1-a, EIF4A, TUB, and RP were the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined that EF1-α, EIF4A, and CYP were stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock protein SHSP, which is related to heat stress in Amorphophallus. In sum, EF1-α and EIF4A were the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different

Real-time holographic endoscopy

Science.gov (United States)

Smigielski, Paul; Albe, Felix; Dischli, Bernard

1992-08-01

Some new experiments concerning holographic endoscopy are presented. The quantitative measurements of deformations of objects are obtained by the double-exposure and double- reference beam method, using either a cw-laser or a pulsed laser. Qualitative experiments using an argon laser with time-average holographic endoscopy are also presented. A video film on real-time endoscopic holographic interferometry was recorded with the help of a frequency-doubled YAG-laser working at 25 Hz for the first time.

MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments

NARCIS (Netherlands)

Bustin, S.A.; Beaulieu, J.F.; Huggett, J.; Jaggi, R.; Kibenge, F.S.; Olsvik, P.A.; Penning, L.C.; Toegel, S.

2010-01-01

MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments Stephen A Bustin1 , Jean-François Beaulieu2 , Jim Huggett3 , Rolf Jaggi4 , Frederick SB Kibenge5 , Pål A Olsvik6 , Louis C Penning7 and Stefan Toegel8 1 Centre for

Chemisorption of iodine-125 to gold nanoparticles allows for real-time quantitation and potential use in nanomedicine

Energy Technology Data Exchange (ETDEWEB)

Walsh, Adrian A, E-mail: a.walsh@nanobiosols.com [Liverpool Science Park, Nano Biosols Ltd (United Kingdom)

2017-04-15

Gold nanoparticles have been available for many years as a research tool in the life sciences due to their electron density and optical properties. New applications are continually being developed, particularly in nanomedicine. One drawback is the need for an easy, real-time quantitation method for gold nanoparticles so that the effects observed in in vitro cell toxicity assays and cell uptake studies can be interpreted quantitatively in terms of nanoparticle loading. One potential method of quantifying gold nanoparticles in real time is by chemisorption of iodine-125, a gamma emitter, to the nanoparticles. This paper revisits the labelling of gold nanoparticles with iodine-125, first described 30 years ago and never fully exploited since. We explore the chemical properties and usefulness in quantifying bio-functionalised gold nanoparticle binding in a quick and simple manner. The gold particles were labelled specifically and quantitatively simply by mixing the two items. The nature of the labelling is chemisorption and is robust, remaining bound over several weeks in a variety of cell culture media. Chemisorption was confirmed as potassium iodide can remove the label whereas sodium chloride and many other buffers had no effect. Particles precoated in polymers or proteins can be labelled just as efficiently allowing for post-labelling experiments in situ rather than using radioactive gold atoms in the production process. We also demonstrate that interparticle exchange of I-125 between different size particles does not appear to take place confirming the affinity of the binding.

Chemisorption of iodine-125 to gold nanoparticles allows for real-time quantitation and potential use in nanomedicine

Science.gov (United States)

Walsh, Adrian A.

2017-04-01

Gold nanoparticles have been available for many years as a research tool in the life sciences due to their electron density and optical properties. New applications are continually being developed, particularly in nanomedicine. One drawback is the need for an easy, real-time quantitation method for gold nanoparticles so that the effects observed in in vitro cell toxicity assays and cell uptake studies can be interpreted quantitatively in terms of nanoparticle loading. One potential method of quantifying gold nanoparticles in real time is by chemisorption of iodine-125, a gamma emitter, to the nanoparticles. This paper revisits the labelling of gold nanoparticles with iodine-125, first described 30 years ago and never fully exploited since. We explore the chemical properties and usefulness in quantifying bio-functionalised gold nanoparticle binding in a quick and simple manner. The gold particles were labelled specifically and quantitatively simply by mixing the two items. The nature of the labelling is chemisorption and is robust, remaining bound over several weeks in a variety of cell culture media. Chemisorption was confirmed as potassium iodide can remove the label whereas sodium chloride and many other buffers had no effect. Particles precoated in polymers or proteins can be labelled just as efficiently allowing for post-labelling experiments in situ rather than using radioactive gold atoms in the production process. We also demonstrate that interparticle exchange of I-125 between different size particles does not appear to take place confirming the affinity of the binding.

Chemisorption of iodine-125 to gold nanoparticles allows for real-time quantitation and potential use in nanomedicine

International Nuclear Information System (INIS)

Walsh, Adrian A

2017-01-01

Gold nanoparticles have been available for many years as a research tool in the life sciences due to their electron density and optical properties. New applications are continually being developed, particularly in nanomedicine. One drawback is the need for an easy, real-time quantitation method for gold nanoparticles so that the effects observed in in vitro cell toxicity assays and cell uptake studies can be interpreted quantitatively in terms of nanoparticle loading. One potential method of quantifying gold nanoparticles in real time is by chemisorption of iodine-125, a gamma emitter, to the nanoparticles. This paper revisits the labelling of gold nanoparticles with iodine-125, first described 30 years ago and never fully exploited since. We explore the chemical properties and usefulness in quantifying bio-functionalised gold nanoparticle binding in a quick and simple manner. The gold particles were labelled specifically and quantitatively simply by mixing the two items. The nature of the labelling is chemisorption and is robust, remaining bound over several weeks in a variety of cell culture media. Chemisorption was confirmed as potassium iodide can remove the label whereas sodium chloride and many other buffers had no effect. Particles precoated in polymers or proteins can be labelled just as efficiently allowing for post-labelling experiments in situ rather than using radioactive gold atoms in the production process. We also demonstrate that interparticle exchange of I-125 between different size particles does not appear to take place confirming the affinity of the binding.

Quantitation of RHD by real-time polymerase chain reaction for determination of RHD zygosity and RHD mosaicism/chimerism

DEFF Research Database (Denmark)

Krog, Grethe Risum; Clausen, Frederik Banch; Dziegiel, Morten Hanefeld

2007-01-01

Determination of RHD zygosity of the spouse is crucial in preconception counseling of families with history of hemolytic disease of the fetus and newborn caused by anti-D. RHD zygosity can be determined by quantitative real-time polymerase chain reaction (PCR) basically by determining RHD dosage,......, and this feature is relevant in investigating RHD mosaicism and chimerism....

Comparative Evaluation of Four Real-Time PCR Methods for the Quantitative Detection of Epstein-Barr Virus from Whole Blood Specimens.

Science.gov (United States)

Buelow, Daelynn; Sun, Yilun; Tang, Li; Gu, Zhengming; Pounds, Stanley; Hayden, Randall

2016-07-01

Monitoring of Epstein-Barr virus (EBV) load in immunocompromised patients has become integral to their care. An increasing number of reagents are available for quantitative detection of EBV; however, there are little published comparative data. Four real-time PCR systems (one using laboratory-developed reagents and three using analyte-specific reagents) were compared with one another for detection of EBV from whole blood. Whole blood specimens seeded with EBV were used to determine quantitative linearity, analytical measurement range, lower limit of detection, and CV for each assay. Retrospective testing of 198 clinical samples was performed in parallel with all methods; results were compared to determine relative quantitative and qualitative performance. All assays showed similar performance. No significant difference was found in limit of detection (3.12-3.49 log10 copies/mL; P = 0.37). A strong qualitative correlation was seen with all assays that used clinical samples (positive detection rates of 89.5%-95.8%). Quantitative correlation of clinical samples across assays was also seen in pairwise regression analysis, with R(2) ranging from 0.83 to 0.95. Normalizing clinical sample results to IU/mL did not alter the quantitative correlation between assays. Quantitative EBV detection by real-time PCR can be performed over a wide linear dynamic range, using three different commercially available reagents and laboratory-developed methods. EBV was detected with comparable sensitivity and quantitative correlation for all assays. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

A machine learning approach for real-time modelling of tissue deformation in image-guided neurosurgery.

Science.gov (United States)

Tonutti, Michele; Gras, Gauthier; Yang, Guang-Zhong

2017-07-01

Accurate reconstruction and visualisation of soft tissue deformation in real time is crucial in image-guided surgery, particularly in augmented reality (AR) applications. Current deformation models are characterised by a trade-off between accuracy and computational speed. We propose an approach to derive a patient-specific deformation model for brain pathologies by combining the results of pre-computed finite element method (FEM) simulations with machine learning algorithms. The models can be computed instantaneously and offer an accuracy comparable to FEM models. A brain tumour is used as the subject of the deformation model. Load-driven FEM simulations are performed on a tetrahedral brain mesh afflicted by a tumour. Forces of varying magnitudes, positions, and inclination angles are applied onto the brain's surface. Two machine learning algorithms-artificial neural networks (ANNs) and support vector regression (SVR)-are employed to derive a model that can predict the resulting deformation for each node in the tumour's mesh. The tumour deformation can be predicted in real time given relevant information about the geometry of the anatomy and the load, all of which can be measured instantly during a surgical operation. The models can predict the position of the nodes with errors below 0.3mm, beyond the general threshold of surgical accuracy and suitable for high fidelity AR systems. The SVR models perform better than the ANN's, with positional errors for SVR models reaching under 0.2mm. The results represent an improvement over existing deformation models for real time applications, providing smaller errors and high patient-specificity. The proposed approach addresses the current needs of image-guided surgical systems and has the potential to be employed to model the deformation of any type of soft tissue. Copyright © 2017 Elsevier B.V. All rights reserved.

Real-time PCR assays for the quantitation of rDNA from apricot and other plant species in marzipan.

Science.gov (United States)

Haase, Ilka; Brüning, Philipp; Matissek, Reinhard; Fischer, Markus

2013-04-10

Marzipan or marzipan raw paste is a typical German sweet which is consumed directly or is used as an ingredient in the bakery industry/confectionery (e.g., in stollen) and as filling for chocolate candies. Almonds (blanched and pealed) and sugar are the only ingredients for marzipan production according to German food guidelines. Especially for the confectionery industry, the use of persipan, which contains apricot or peach kernels instead of almonds, is preferred due to its stronger aroma. In most of the companies, both raw pastes are produced, in most cases on the same production line, running the risk of an unintended cross contamination. Additionally, due to high almond market values, dilutions of marzipan with cheaper seeds may occur. Especially in the case of apricot and almond, the close relationship of both species is a challenge for the analysis. DNA based methods for the qualitative detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea in marzipan have recently been published. In this study, different quantitation strategies on the basis of real-time PCR have been evaluated and a relative quantitation method with a reference amplification product was shown to give the best results. As the real-time PCR is based on the high copy rDNA-cluster, even contaminations <1% can be reliably quantitated.

Rapid real-time PCR assay for culture and tissue identification of Geomyces destructans: the etiologic agent of bat geomycosis (white nose syndrome).

Science.gov (United States)

Chaturvedi, Sudha; Rudd, Robert J; Davis, April; Victor, Tanya R; Li, Xiaojiang; Appler, Kim A; Rajkumar, Sunanda S; Chaturvedi, Vishnu

2011-10-01

Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS-PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10(-1)-5 × 10(7)), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)-PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.

Real-time subject-specific monitoring of internal deformations and stresses in the soft tissues of the foot: a new approach in gait analysis.

Science.gov (United States)

Yarnitzky, G; Yizhar, Z; Gefen, A

2006-01-01

No technology is presently available to provide real-time information on internal deformations and stresses in plantar soft tissues of individuals during evaluation of the gait pattern. Because internal deformations and stresses in the plantar pad are critical factors in foot injuries such as diabetic foot ulceration, this severely limits evaluation of patients. To allow such real-time subject-specific analysis, we developed a hierarchal modeling system which integrates a two-dimensional gross structural model of the foot (high-order model) with local finite element (FE) models of the plantar tissue padding the calcaneus and medial metatarsal heads (low-order models). The high-order whole-foot model provides real-time analytical evaluations of the time-dependent plantar fascia tensile forces during the stance phase. These force evaluations are transferred, together with foot-shoe local reaction forces, also measured in real time (under the calcaneus, medial metatarsals and hallux), to the low-order FE models of the plantar pad, where they serve as boundary conditions for analyses of local deformations and stresses in the plantar pad. After careful verification of our custom-made FE solver and of our foot model system with respect to previous literature and against experimental results from a synthetic foot phantom, we conducted human studies in which plantar tissue loading was evaluated in real time during treadmill gait in healthy individuals (N = 4). We concluded that internal deformations and stresses in the plantar pad during gait cannot be predicted from merely measuring the foot-shoe force reactions. Internal loading of the plantar pad is constituted by a complex interaction between the anatomical structure and mechanical behavior of the foot skeleton and soft tissues, the body characteristics, the gait pattern and footwear. Real-time FE monitoring of internal deformations and stresses in the plantar pad is therefore required to identify elevated deformation

WetLab-2: Tools for Conducting On-Orbit Quantitative Real-Time Gene Expression Analysis on ISS

Science.gov (United States)

Parra, Macarena; Almeida, Eduardo; Boone, Travis; Jung, Jimmy; Schonfeld, Julie

2014-01-01

The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR system, the Cepheid SmartCycler and will fly it in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid ramp times and the ability to detect up to four separate fluorescent channels at one time enabling multiplex assays that can be used for normalization and to study multiple genes of interest in each module. The team is currently working with Cepheid to enable the downlink of data from the ISS to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project has adapted commercial technology to design a module that can lyse cells and extract RNA of sufficient quality and quantity for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. The WetLab-2 system is capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experiment parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Researchers will also be able to sample multigenerational changes in organisms. Finally, the system can be

Real-time three-dimensional ultrasound-assisted axillary plexus block defines soft tissue planes.

Science.gov (United States)

Clendenen, Steven R; Riutort, Kevin; Ladlie, Beth L; Robards, Christopher; Franco, Carlo D; Greengrass, Roy A

2009-04-01

Two-dimensional (2D) ultrasound is commonly used for regional block of the axillary brachial plexus. In this technical case report, we described a real-time three-dimensional (3D) ultrasound-guided axillary block. The difference between 2D and 3D ultrasound is similar to the difference between plain radiograph and computer tomography. Unlike 2D ultrasound that captures a planar image, 3D ultrasound technology acquires a 3D volume of information that enables multiple planes of view by manipulating the image without movement of the ultrasound probe. Observation of the brachial plexus in cross-section demonstrated distinct linear hyperechoic tissue structures (loose connective tissue) that initially inhibited the flow of the local anesthesia. After completion of the injection, we were able to visualize the influence of arterial pulsation on the spread of the local anesthesia. Possible advantages of this novel technology over current 2D methods are wider image volume and the capability to manipulate the planes of the image without moving the probe.

Integrating viscoelastic mass spring dampers into position-based dynamics to simulate soft tissue deformation in real time.

Science.gov (United States)

Xu, Lang; Lu, Yuhua; Liu, Qian

2018-02-01

We propose a novel method to simulate soft tissue deformation for virtual surgery applications. The method considers the mechanical properties of soft tissue, such as its viscoelasticity, nonlinearity and incompressibility; its speed, stability and accuracy also meet the requirements for a surgery simulator. Modifying the traditional equation for mass spring dampers (MSD) introduces nonlinearity and viscoelasticity into the calculation of elastic force. Then, the elastic force is used in the constraint projection step for naturally reducing constraint potential. The node position is enforced by the combined spring force and constraint conservative force through Newton's second law. We conduct a comparison study of conventional MSD and position-based dynamics for our new integrating method. Our approach enables stable, fast and large step simulation by freely controlling visual effects based on nonlinearity, viscoelasticity and incompressibility. We implement a laparoscopic cholecystectomy simulator to demonstrate the practicality of our method, in which liver and gallbladder deformation can be simulated in real time. Our method is an appropriate choice for the development of real-time virtual surgery applications.

Two-dimensional and 3-D images of thick tissue using time-constrained times-of-flight and absorbance spectrophotometry

Science.gov (United States)

Benaron, David A.; Lennox, M.; Stevenson, David K.

1992-05-01

assessment. The technique may lead to real-time optical imaging and quantitation of tissues oxygen delivery.

Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.

Directory of Open Access Journals (Sweden)

Pengfei Lin

Full Text Available The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS, NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.

Real-time quasi-3D tomographic reconstruction

Science.gov (United States)

Buurlage, Jan-Willem; Kohr, Holger; Palenstijn, Willem Jan; Joost Batenburg, K.

2018-06-01

Developments in acquisition technology and a growing need for time-resolved experiments pose great computational challenges in tomography. In addition, access to reconstructions in real time is a highly demanded feature but has so far been out of reach. We show that by exploiting the mathematical properties of filtered backprojection-type methods, having access to real-time reconstructions of arbitrarily oriented slices becomes feasible. Furthermore, we present , software for visualization and on-demand reconstruction of slices. A user of can interactively shift and rotate slices in a GUI, while the software updates the slice in real time. For certain use cases, the possibility to study arbitrarily oriented slices in real time directly from the measured data provides sufficient visual and quantitative insight. Two such applications are discussed in this article.

Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station.

Directory of Open Access Journals (Sweden)

Macarena Parra

Full Text Available The International Space Station (ISS National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for

Real-time PCR in virology.

Science.gov (United States)

Mackay, Ian M; Arden, Katherine E; Nitsche, Andreas

2002-03-15

The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

Verus: A Tool for Quantitative Analysis of Finite-State Real-Time Systems.

Science.gov (United States)

1996-08-12

Symbolic model checking is a technique for verifying finite-state concurrent systems that has been extended to handle real - time systems . Models with...up to 10(exp 30) states can often be verified in minutes. In this paper, we present a new tool to analyze real - time systems , based on this technique...We have designed a language, called Verus, for the description of real - time systems . Such a description is compiled into a state-transition graph and

Species identification of Cannabis sativa using real-time quantitative PCR (qPCR).

Science.gov (United States)

Johnson, Christopher E; Premasuthan, Amritha; Satkoski Trask, Jessica; Kanthaswamy, Sree

2013-03-01

Most narcotics-related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3' exon-trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real-time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa. © 2013 American Academy of Forensic Sciences.

Quantitative Microbiological Study of Human Carious Dentine by Culture and Real-Time PCR: Association of Anaerobes with Histopathological Changes in Chronic Pulpitis

Science.gov (United States)

Martin, F. Elizabeth; Nadkarni, Mangala A.; Jacques, Nicholas A.; Hunter, Neil

2002-01-01

The bacteria found in carious dentine were correlated with the tissue response of the dental pulps of 65 teeth extracted from patients with advanced caries and pulpitis. Standardized homogenates of carious dentine were plated onto selective and nonselective media under anaerobic and microaerophilic conditions. In addition, real-time PCR was used to quantify the recovery of anaerobic bacteria. Primers and fluorogenic probes were designed to detect the total anaerobic microbial load, the genera Prevotella and Fusobacterium, and the species Prevotella melaninogenica, Porphyromonas endodontalis, Porphyromonas gingivalis, and Micromonas (formerly Peptostreptococcus) micros. The pulpal pathology was categorized according to the cellular response and degenerative changes. Analysis of cultured bacteria showed a predominance of gram-positive microorganisms, particularly lactobacilli. Gram-negative bacteria were also present in significant numbers with Prevotella spp., the most numerous anaerobic group cultured. Real-time PCR analysis indicated a greater microbial load than that determined by colony counting. The total number of anaerobes detected was 41-fold greater by real-time PCR than by colony counting, while the numbers of Prevotella and Fusobacterium spp. detected were 82- and 2.4-fold greater by real-time PCR than by colony counting, respectively. Real-time PCR also identified M. micros, P. endodontalis, and P. gingivalis in 71, 60, and 52% of carious samples, respectively. Correlation matrices of the real-time PCR data revealed significant positive associations between M. micros and P. endodontalis detection and inflammatory degeneration of pulpal tissues. These anaerobes have been strongly implicated in endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of high levels of these bacteria in carious lesions may be indicative of irreversible pulpal pathology. PMID:11980945

Reference Gene Screening for Analyzing Gene Expression Across Goat Tissue

Directory of Open Access Journals (Sweden)

Yu Zhang

2013-12-01

Full Text Available Real-time quantitative PCR (qRT-PCR is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2 in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.

Real-time dynamic imaging of virus distribution in vivo.

Directory of Open Access Journals (Sweden)

Sean E Hofherr

2011-02-01

Full Text Available The distribution of viruses and gene therapy vectors is difficult to assess in a living organism. For instance, trafficking in murine models can usually only be assessed after sacrificing the animal for tissue sectioning or extraction. These assays are laborious requiring whole animal sectioning to ascertain tissue localization. They also obviate the ability to perform longitudinal or kinetic studies in one animal. To track viruses after systemic infection, we have labeled adenoviruses with a near-infrared (NIR fluorophore and imaged these after intravenous injection in mice. Imaging was able to track and quantitate virus particles entering the jugular vein simultaneous with injection, appearing in the heart within 500 milliseconds, distributing in the bloodstream and throughout the animal within 7 seconds, and that the bulk of virus distribution was essentially complete within 3 minutes. These data provide the first in vivo real-time tracking of the rapid initial events of systemic virus infection.

A Human Fecal Contamination Score for Ranking Recreational Sites using the HF183/BacR287 Quantitative Real-Time PCR Method

Science.gov (United States)

Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and manag...

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)

Science.gov (United States)

Quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technique for measuring gene expression levels due to its simplicity, specificity, and sensitivity. Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a...

Development of a real-time PCR to detect Demodex canis DNA in different tissue samples.

Science.gov (United States)

Ravera, Ivan; Altet, Laura; Francino, Olga; Bardagí, Mar; Sánchez, Armand; Ferrer, Lluís

2011-02-01

The present study reports the development of a real-time polymerase chain reaction (PCR) to detect Demodex canis DNA on different tissue samples. The technique amplifies a 166 bp of D. canis chitin synthase gene (AB 080667) and it has been successfully tested on hairs extracted with their roots and on formalin-fixed paraffin embedded skin biopsies. The real-time PCR amplified on the hairs of all 14 dogs with a firm diagnosis of demodicosis and consistently failed to amplify on negative controls. Eleven of 12 skin biopsies with a morphologic diagnosis of canine demodicosis were also positive. Sampling hairs on two skin points (lateral face and interdigital skin), D. canis DNA was detected on nine of 51 healthy dogs (17.6%) a much higher percentage than previously reported with microscopic studies. Furthermore, it is foreseen that if the number of samples were increased, the percentage of positive dogs would probably also grow. Moreover, in four of the six dogs with demodicosis, the samples taken from non-lesioned skin were positive. This finding, if confirmed in further studies, suggests that demodicosis is a generalized phenomenon in canine skin, due to proliferation of local mite populations, even though macroscopic lesions only appear in certain areas. The real-time PCR technique to detect D. canis DNA described in this work is a useful tool to advance our understanding of canine demodicosis.

Comparison of three-way and four-way calibration for the real-time quantitative analysis of drug hydrolysis in complex dynamic samples by excitation-emission matrix fluorescence

Science.gov (United States)

Yin, Xiao-Li; Gu, Hui-Wen; Liu, Xiao-Lu; Zhang, Shan-Hui; Wu, Hai-Long

2018-03-01

Multiway calibration in combination with spectroscopic technique is an attractive tool for online or real-time monitoring of target analyte(s) in complex samples. However, how to choose a suitable multiway calibration method for the resolution of spectroscopic-kinetic data is a troubling problem in practical application. In this work, for the first time, three-way and four-way fluorescence-kinetic data arrays were generated during the real-time monitoring of the hydrolysis of irinotecan (CPT-11) in human plasma by excitation-emission matrix fluorescence. Alternating normalization-weighted error (ANWE) and alternating penalty trilinear decomposition (APTLD) were used as three-way calibration for the decomposition of the three-way kinetic data array, whereas alternating weighted residual constraint quadrilinear decomposition (AWRCQLD) and alternating penalty quadrilinear decomposition (APQLD) were applied as four-way calibration to the four-way kinetic data array. The quantitative results of the two kinds of calibration models were fully compared from the perspective of predicted real-time concentrations, spiked recoveries of initial concentration, and analytical figures of merit. The comparison study demonstrated that both three-way and four-way calibration models could achieve real-time quantitative analysis of the hydrolysis of CPT-11 in human plasma under certain conditions. However, it was also found that both of them possess some critical advantages and shortcomings during the process of dynamic analysis. The conclusions obtained in this paper can provide some helpful guidance for the reasonable selection of multiway calibration models to achieve the real-time quantitative analysis of target analyte(s) in complex dynamic systems.

Validation of Reference Genes for Quantitative Expression Analysis by Real-Time RT-PCR in Four Lepidopteran Insects

OpenAIRE

Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei

2012-01-01

Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae...

Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera

Directory of Open Access Journals (Sweden)

Amiteye Samuel

2011-08-01

Full Text Available Abstract Background Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates. Results Based on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18, Efalpha1 (Elongation factor 1 alpha, ACT 2 (Actin2, UBQ (polyubiquitin, PEX4 (Peroxisomal ubiquitin conjugating enzyme and At1g09770.1 (Arabidopsis thaliana cell division cycle 5. Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEfα1 or BoechUBQ and BoechEfα1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed

Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods.

Science.gov (United States)

Edagawa, Akiko; Kimura, Akio; Kawabuchi-Kurata, Takako; Adachi, Shinichi; Furuhata, Katsunori; Miyamoto, Hiroshi

2015-10-19

We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

Detection of Ophiocordyceps sinensis in soil by quantitative real-time PCR.

Science.gov (United States)

Peng, Qingyun; Zhong, Xin; Lei, Wei; Zhang, Guren; Liu, Xin

2013-03-01

Ophiocordyceps sinensis, one of the best known entomopathogenic fungi in traditional Chinese medicine, parasitizes larvae of the moth genus Thitarodes, which lives in soil tunnels. However, little is known about the spatial distribution of O. sinensis in the soil. We established a protocol for DNA extraction, purification, and quantification of O. sinensis in soil with quantitative real-time PCR targeting the internal transcribed spacer region. The method was assessed using 34 soil samples from Tibet. No inhibitory effects in purified soil DNA extracts were detected. The standard curve method for absolute DNA quantification generated crossing point values that were strongly and linearly correlated to the log10 of the initial amount of O. sinensis genomic DNA (r(2) = 0.999) over 7 orders of magnitude (4 × 10(1) to 4 × 10(7) fg). The amplification efficiency and y-intercept value of the standard curve were 1.953 and 37.70, respectively. The amount of O. sinensis genomic DNA decreased with increasing soil depth and horizontal distance from a sclerotium (P protocol is rapid, specific, sensitive, and provides a powerful tool for quantification of O. sinensis from soil.

Quantitative Evaluation of Fire and EMS Mobilization Times

CERN Document Server

Upson, Robert

2010-01-01

Quantitative Evaluation of Fire and EMS Mobilization Times presents comprehensive empirical data on fire emergency and EMS call processing and turnout times, and aims to improve the operational benchmarks of NFPA peer consensus standards through a close examination of real-world data. The book also identifies and analyzes the elements that can influence EMS mobilization response times. Quantitative Evaluation of Fire and EMS Mobilization Times is intended for practitioners as a tool for analyzing fire emergency response times and developing methods for improving them. Researchers working in a

[Real time 3D echocardiography

Science.gov (United States)

Bauer, F.; Shiota, T.; Thomas, J. D.

2001-01-01

Three-dimensional representation of the heart is an old concern. Usually, 3D reconstruction of the cardiac mass is made by successive acquisition of 2D sections, the spatial localisation and orientation of which require complex guiding systems. More recently, the concept of volumetric acquisition has been introduced. A matricial emitter-receiver probe complex with parallel data processing provides instantaneous of a pyramidal 64 degrees x 64 degrees volume. The image is restituted in real time and is composed of 3 planes (planes B and C) which can be displaced in all spatial directions at any time during acquisition. The flexibility of this system of acquisition allows volume and mass measurement with greater accuracy and reproducibility, limiting inter-observer variability. Free navigation of the planes of investigation allows reconstruction for qualitative and quantitative analysis of valvular heart disease and other pathologies. Although real time 3D echocardiography is ready for clinical usage, some improvements are still necessary to improve its conviviality. Then real time 3D echocardiography could be the essential tool for understanding, diagnosis and management of patients.

Real-time estimation of lesion depth and control of radiofrequency ablation within ex vivo animal tissues using a neural network.

Science.gov (United States)

Wang, Yearnchee Curtis; Chan, Terence Chee-Hung; Sahakian, Alan Varteres

2018-01-04

Radiofrequency ablation (RFA), a method of inducing thermal ablation (cell death), is often used to destroy tumours or potentially cancerous tissue. Current techniques for RFA estimation (electrical impedance tomography, Nakagami ultrasound, etc.) require long compute times (≥ 2 s) and measurement devices other than the RFA device. This study aims to determine if a neural network (NN) can estimate ablation lesion depth for control of bipolar RFA using complex electrical impedance - since tissue electrical conductivity varies as a function of tissue temperature - in real time using only the RFA therapy device's electrodes. Three-dimensional, cubic models comprised of beef liver, pork loin or pork belly represented target tissue. Temperature and complex electrical impedance from 72 data generation ablations in pork loin and belly were used for training the NN (403 s on Xeon processor). NN inputs were inquiry depth, starting complex impedance and current complex impedance. Training-validation-test splits were 70%-0%-30% and 80%-10%-10% (overfit test). Once the NN-estimated lesion depth for a margin reached the target lesion depth, RFA was stopped for that margin of tissue. The NN trained to 93% accuracy and an NN-integrated control ablated tissue to within 1.0 mm of the target lesion depth on average. Full 15-mm depth maps were calculated in 0.2 s on a single-core ARMv7 processor. The results show that a NN could make lesion depth estimations in real-time using less in situ devices than current techniques. With the NN-based technique, physicians could deliver quicker and more precise ablation therapy.

Statistical aspects of quantitative real-time PCR experiment design

Czech Academy of Sciences Publication Activity Database

Kitchen, R.R.; Kubista, Mikael; Tichopád, Aleš

2010-01-01

Roč. 50, č. 4 (2010), s. 231-236 ISSN 1046-2023 R&D Projects: GA AV ČR IAA500520809 Institutional research plan: CEZ:AV0Z50520701 Keywords : Real-time PCR * Experiment design * Nested analysis of variance Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.527, year: 2010

Real time sensor for therapeutic radiation delivery

International Nuclear Information System (INIS)

Bliss, M.; Craig, R.A.; Reeder, P.L.

1998-01-01

The invention is a real time sensor for therapeutic radiation. A probe is placed in or near the patient that senses in real time the dose at the location of the probe. The strength of the dose is determined by either an insertion or an exit probe. The location is determined by a series of vertical and horizontal sensing elements that gives the operator a real time read out dose location relative to placement of the patient. The increased accuracy prevents serious tissue damage to the patient by preventing overdose or delivery of a dose to a wrong location within the body. 14 figs

Comparison of three-way and four-way calibration for the real-time quantitative analysis of drug hydrolysis in complex dynamic samples by excitation-emission matrix fluorescence.

Science.gov (United States)

Yin, Xiao-Li; Gu, Hui-Wen; Liu, Xiao-Lu; Zhang, Shan-Hui; Wu, Hai-Long

2018-03-05

Multiway calibration in combination with spectroscopic technique is an attractive tool for online or real-time monitoring of target analyte(s) in complex samples. However, how to choose a suitable multiway calibration method for the resolution of spectroscopic-kinetic data is a troubling problem in practical application. In this work, for the first time, three-way and four-way fluorescence-kinetic data arrays were generated during the real-time monitoring of the hydrolysis of irinotecan (CPT-11) in human plasma by excitation-emission matrix fluorescence. Alternating normalization-weighted error (ANWE) and alternating penalty trilinear decomposition (APTLD) were used as three-way calibration for the decomposition of the three-way kinetic data array, whereas alternating weighted residual constraint quadrilinear decomposition (AWRCQLD) and alternating penalty quadrilinear decomposition (APQLD) were applied as four-way calibration to the four-way kinetic data array. The quantitative results of the two kinds of calibration models were fully compared from the perspective of predicted real-time concentrations, spiked recoveries of initial concentration, and analytical figures of merit. The comparison study demonstrated that both three-way and four-way calibration models could achieve real-time quantitative analysis of the hydrolysis of CPT-11 in human plasma under certain conditions. However, it was also found that both of them possess some critical advantages and shortcomings during the process of dynamic analysis. The conclusions obtained in this paper can provide some helpful guidance for the reasonable selection of multiway calibration models to achieve the real-time quantitative analysis of target analyte(s) in complex dynamic systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and validation of reference genes for quantitative real-time PCR in Drosophila suzukii (Diptera: Drosophilidae.

Directory of Open Access Journals (Sweden)

Yifan Zhai

Full Text Available To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR data, normalization relative to reliable reference gene(s is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin, were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population, and abiotic (photoperiod, temperature conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper and one web-based comprehensive tool (RefFinder were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.

REAL-TIME ASSESSMENT OF MICROWAVE ABLATION EFFICACY BY NIR SPECTROSCOPIC TECHNIQUE

Directory of Open Access Journals (Sweden)

JINZHE ZHAO

2014-01-01

Full Text Available Microwave ablation (MWA status monitoring in real time plays a key role in assessment of therapeutic effectiveness. As a novel real-time assessment method, near infrared spectroscopy (NIRs was used to evaluate the ablation efficacy. MWA experiments were carried out on in vitro porcine livers. An optical measurement system for biological tissue is developed by our lab to monitor reduced scattering coefficient $(\\mu_{s}^{'}$ at 690 nm of the coagulation zones. It is noted that $\\mu_{s}^{'}$ of liver tissue, which increases as the liver tissue being ablated, is clearly related with the coagulation status. $\\mu_{s}^{'}$ of normal tissue and coagulated tissue is 3–5 and 17–19 cm-1, respectively. Continuous changes of $\\mu_{s}^{'}$ demonstrate that optical parameter can be used as an efficacy evaluation factor because it essentially indicates the degree of thermal damage. Compared with temperature, optical parameter is more sensitive and accurate, which is promising for real-time therapeutic efficacy assessment in MWA.

Fast real-time polymerase chain reaction for quantitative detection of Lactobacillus delbrueckii bacteriophages in milk.

Science.gov (United States)

Martín, Maria Cruz; del Rio, Beatriz; Martínez, Noelia; Magadán, Alfonso H; Alvarez, Miguel A

2008-12-01

One of the main microbiological problems of the dairy industry is the susceptibility of starter bacteria to virus infections. Lactobacillus delbrueckii, a component of thermophilic starter cultures used in the manufacture of several fermented dairy products, including yogurt, is also sensitive to bacteriophage attacks. To avoid the problems associated with these viruses, quick and sensitive detection methods are necessary. In the present study, a fast real-time quantitative polymerase chain reaction assay for the direct detection and quantification of L. delbrueckii phages in milk was developed. A set of primers and a TaqMan MGB probe was designed, based on the lysin gene sequence of different L. delbrueckii phages. The results show the proposed method to be a rapid (total processing time 30 min), specific and highly sensitive technique for detecting L. delbrueckii phages in milk.

Investigations on abundance and activity of microbial sponge symbionts using quantitative real - time PCR

DEFF Research Database (Denmark)

Kumala, Lars; Hentschel, Ute; Bayer, Kristina

Marine sponges are hosts to dense and diverse microbial consortia that are likely to play a key role in the metabolic processes of the host sponge due to their enormous abundance. Common symbioses between nitrogen transforming microorganisms and sponges indicate complex nitrogen cycling within...... the host. Of particular interest is determining the community structure and function of microbial symbionts in order to gain deeper insight into host-symbiont interactions. We investigated the abundance and activity of microbial symbionts in two Mediterranean sponge species using quantitative real-time PCR....... An absolute quantification of functional genes and transcripts in archaeal and bacterial symbionts was conducted to determine their involvement in nitrification and denitrification, comparing the low microbial abundance (LMA) sponge Dysidea avara with the high microbial abundance (HMA) representative Aplysina...

Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods

Directory of Open Access Journals (Sweden)

Akiko Edagawa

2015-10-01

Full Text Available We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR, and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%. Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%. In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8% compared with real-time qPCR alone (46/68, 67.6%. Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1% compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%. Legionella was not detected in the remaining six samples (6/68, 8.8%, irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

POD for Real-Time Simulation of Hyperelastic Soft Biological Tissue Using the Point Collocation Method of Finite Spheres

Directory of Open Access Journals (Sweden)

Suleiman Banihani

2013-01-01

Full Text Available The point collocation method of finite spheres (PCMFS is used to model the hyperelastic response of soft biological tissue in real time within the framework of virtual surgery simulation. The proper orthogonal decomposition (POD model order reduction (MOR technique was used to achieve reduced-order model of the problem, minimizing computational cost. The PCMFS is a physics-based meshfree numerical technique for real-time simulation of surgical procedures where the approximation functions are applied directly on the strong form of the boundary value problem without the need for integration, increasing computational efficiency. Since computational speed has a significant role in simulation of surgical procedures, the proposed technique was able to model realistic nonlinear behavior of organs in real time. Numerical results are shown to demonstrate the effectiveness of the new methodology through a comparison between full and reduced analyses for several nonlinear problems. It is shown that the proposed technique was able to achieve good agreement with the full model; moreover, the computational and data storage costs were significantly reduced.

SU-E-J-197: A Novel Optical Interstitial Fiber Spectroscopic System for Real-Time Tissue Micro-Vascular Hemodynamics Monitoring.

Science.gov (United States)

Zhao, D; Campos, D; Yan, Y; Kimple, R; Jacques, S; van der Kogel, A; Kissick, M

2012-06-01

To demonstrate a novel interstitial optical fiber spectroscopic system, based on diffuse optical spectroscopies with spectral fitting, for the simultaneous monitoring of tumor blood volume and oxygen tension. The technique provides real-time, minimally-invasive and quantification of tissue micro-vascular hemodynamics. An optical fiber prototype probe characterizesthe optical transport in tissue between two large Numerical Aperture (NA) fibers of 200μm core diameter (BFH37-200, ThorLabs) spaced 3-mm apart. Two 21-Ga medical needles are used to protect fiber ends and to facilitate tissue penetration with minimum local blunt trauma in nude mice with xenografts. A 20W white light source (HL-2000-HP, Ocean Optics) is coupled to one fiber with SMA adapter. The other fiber is used to collect light, which is coupled into the spectrometer (QE65000 with Spectrasuite Operating software and OmniDriver, Ocean Optics). The wavelength response of the probe depends on the wavelength dependence of the light source, and of the light signal collection that includes considerable scatter, modeled with Monte-Carlo techniques (S. Jacques 2010 J. of Innov. Opt. Health Sci. 2 123-9). Measured spectra of tissue are normalized by a measured spectrum of a white standard, yielding the transmission spectrum. A head-and-neck xenograft on the flank of a live mouse is used for development. The optical fiber probe delivers and collects light at an arbitrary depth in the tumor. By spectral fitting of the measured transmission spectrum, an analysis of blood volume and oxygen tension is obtained from the fitting parameters in real time. A newly developed optical fiber spectroscopic system with an optical fiber probe takes spectroscopic techniques to a much deeper level in a tumor, which has potential applications for real-time monitoring hypoxic cell population dynamics for an eventual adaptive therapy metric of particular use in hypofractionated radiotherapy. © 2012 American Association of

Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis.

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Peeters, M; Huang, C L; Vonk, L A; Lu, Z F; Bank, R A; Helder, M N; Doulabi, B Zandieh

2016-11-01

Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised.Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016

Quantitative characterization of viscoelastic behavior in tissue-mimicking phantoms and ex vivo animal tissues.

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Ashkan Maccabi

Full Text Available Viscoelasticity of soft tissue is often related to pathology, and therefore, has become an important diagnostic indicator in the clinical assessment of suspect tissue. Surgeons, particularly within head and neck subsites, typically use palpation techniques for intra-operative tumor detection. This detection method, however, is highly subjective and often fails to detect small or deep abnormalities. Vibroacoustography (VA and similar methods have previously been used to distinguish tissue with high-contrast, but a firm understanding of the main contrast mechanism has yet to be verified. The contributions of tissue mechanical properties in VA images have been difficult to verify given the limited literature on viscoelastic properties of various normal and diseased tissue. This paper aims to investigate viscoelasticity theory and present a detailed description of viscoelastic experimental results obtained in tissue-mimicking phantoms (TMPs and ex vivo tissues to verify the main contrast mechanism in VA and similar imaging modalities. A spherical-tip micro-indentation technique was employed with the Hertzian model to acquire absolute, quantitative, point measurements of the elastic modulus (E, long term shear modulus (η, and time constant (τ in homogeneous TMPs and ex vivo tissue in rat liver and porcine liver and gallbladder. Viscoelastic differences observed between porcine liver and gallbladder tissue suggest that imaging modalities which utilize the mechanical properties of tissue as a primary contrast mechanism can potentially be used to quantitatively differentiate between proximate organs in a clinical setting. These results may facilitate more accurate tissue modeling and add information not currently available to the field of systems characterization and biomedical research.

Quantitative characterization of viscoelastic behavior in tissue-mimicking phantoms and ex vivo animal tissues.

Science.gov (United States)

Maccabi, Ashkan; Shin, Andrew; Namiri, Nikan K; Bajwa, Neha; St John, Maie; Taylor, Zachary D; Grundfest, Warren; Saddik, George N

2018-01-01

Viscoelasticity of soft tissue is often related to pathology, and therefore, has become an important diagnostic indicator in the clinical assessment of suspect tissue. Surgeons, particularly within head and neck subsites, typically use palpation techniques for intra-operative tumor detection. This detection method, however, is highly subjective and often fails to detect small or deep abnormalities. Vibroacoustography (VA) and similar methods have previously been used to distinguish tissue with high-contrast, but a firm understanding of the main contrast mechanism has yet to be verified. The contributions of tissue mechanical properties in VA images have been difficult to verify given the limited literature on viscoelastic properties of various normal and diseased tissue. This paper aims to investigate viscoelasticity theory and present a detailed description of viscoelastic experimental results obtained in tissue-mimicking phantoms (TMPs) and ex vivo tissues to verify the main contrast mechanism in VA and similar imaging modalities. A spherical-tip micro-indentation technique was employed with the Hertzian model to acquire absolute, quantitative, point measurements of the elastic modulus (E), long term shear modulus (η), and time constant (τ) in homogeneous TMPs and ex vivo tissue in rat liver and porcine liver and gallbladder. Viscoelastic differences observed between porcine liver and gallbladder tissue suggest that imaging modalities which utilize the mechanical properties of tissue as a primary contrast mechanism can potentially be used to quantitatively differentiate between proximate organs in a clinical setting. These results may facilitate more accurate tissue modeling and add information not currently available to the field of systems characterization and biomedical research.

On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

Science.gov (United States)

Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Tran, Luan; Schonfeld, Julie

2015-01-01

NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of

A quantitative method to evaluate mesenchymal stem cell lipofection using real-time PCR.

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Ribeiro, S C; Mendes, R; Madeira, C; Monteiro, G A; da Silva, C L; Cabral, J M S

2010-01-01

Genetic modification of human mesenchymal stem cells (MSC) is a powerful tool to improve the therapeutic utility of these cells and to increase the knowledge on their regulation mechanisms. In this context, strong efforts have been made recently to develop efficient nonviral gene delivery systems. Although several studies addressed this question most of them use the end product of a reporter gene instead of the DNA uptake quantification to test the transfection efficiency. In this study, we established a method based on quantitative real-time PCR (RT-PCR) to determine the intracellular plasmid DNA copy number in human MSC after lipofection. The procedure requires neither specific cell lysis nor DNA purification. The influence of cell number on the RT-PCR sensitivity was evaluated. The method showed good reproducibility, high sensitivity, and a wide linear range of 75-2.5 x 10⁶ plasmid DNA copies per cell. RT-PCR results were then compared with the percentage of transfected cells assessed by flow cytometry analysis, which showed that flow cytometry-based results are not always proportional to plasmid cellular uptake determined by RT-PCR. This work contributed for the establishment of a rapid quantitative assay to determine intracellular plasmid DNA in stem cells, which will be extremely beneficial for the optimization of gene delivery strategies. © 2010 American Institute of Chemical Engineers

A finite element-based machine learning approach for modeling the mechanical behavior of the breast tissues under compression in real-time.

Science.gov (United States)

Martínez-Martínez, F; Rupérez-Moreno, M J; Martínez-Sober, M; Solves-Llorens, J A; Lorente, D; Serrano-López, A J; Martínez-Sanchis, S; Monserrat, C; Martín-Guerrero, J D

2017-11-01

This work presents a data-driven method to simulate, in real-time, the biomechanical behavior of the breast tissues in some image-guided interventions such as biopsies or radiotherapy dose delivery as well as to speed up multimodal registration algorithms. Ten real breasts were used for this work. Their deformation due to the displacement of two compression plates was simulated off-line using the finite element (FE) method. Three machine learning models were trained with the data from those simulations. Then, they were used to predict in real-time the deformation of the breast tissues during the compression. The models were a decision tree and two tree-based ensemble methods (extremely randomized trees and random forest). Two different experimental setups were designed to validate and study the performance of these models under different conditions. The mean 3D Euclidean distance between nodes predicted by the models and those extracted from the FE simulations was calculated to assess the performance of the models in the validation set. The experiments proved that extremely randomized trees performed better than the other two models. The mean error committed by the three models in the prediction of the nodal displacements was under 2 mm, a threshold usually set for clinical applications. The time needed for breast compression prediction is sufficiently short to allow its use in real-time (<0.2 s). Copyright © 2017 Elsevier Ltd. All rights reserved.

QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME

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Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.ABSTRACTDNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or du...

Real-time three-dimensional soft tissue reconstruction for laparoscopic surgery.

Science.gov (United States)

Kowalczuk, Jędrzej; Meyer, Avishai; Carlson, Jay; Psota, Eric T; Buettner, Shelby; Pérez, Lance C; Farritor, Shane M; Oleynikov, Dmitry

2012-12-01

Accurate real-time 3D models of the operating field have the potential to enable augmented reality for endoscopic surgery. A new system is proposed to create real-time 3D models of the operating field that uses a custom miniaturized stereoscopic video camera attached to a laparoscope and an image-based reconstruction algorithm implemented on a graphics processing unit (GPU). The proposed system was evaluated in a porcine model that approximates the viewing conditions of in vivo surgery. To assess the quality of the models, a synthetic view of the operating field was produced by overlaying a color image on the reconstructed 3D model, and an image rendered from the 3D model was compared with a 2D image captured from the same view. Experiments conducted with an object of known geometry demonstrate that the system produces 3D models accurate to within 1.5 mm. The ability to produce accurate real-time 3D models of the operating field is a significant advancement toward augmented reality in minimally invasive surgery. An imaging system with this capability will potentially transform surgery by helping novice and expert surgeons alike to delineate variance in internal anatomy accurately.

Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

Science.gov (United States)

2014-01-01

Background Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. Results A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual. Conclusions The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level

[Usefulness of a real-time quantitative polymerase-chain reaction (PCR) assay for the diagnosis of congenital and postnatal cytomegalovirus infection].

Science.gov (United States)

Reina, J; Weber, I; Riera, E; Busquets, M; Morales, C

2014-05-01

Cytomegalovirus (CMV) is the main virus causing congenital and postnatal infections in the pediatric population. The aim of this study is to evaluate the usefulness of a quantitative real-time PCR in the diagnosis of these infections using urine as a single sample. We studied all the urine samples of newborns (< 7 days) with suspected congenital infection, and urine of patients with suspected postnatal infection (urine negative at birth). Urines were simultaneously studied by cell culture, qualitative PCR (PCRc), and quantitative real-time PCR (PCRq). We analyzed 332 urine samples (270 to rule out congenital infection and 62 postnatal infections). Of the first, 22 were positive in the PCRq, 19 in the PCRc, and 17 in the culture. PCRq had a sensitivity of 100%, on comparing the culture with the rest of the techniques. Using the PCRq as a reference method, culture had a sensitivity of 77.2%, and PCRc 86.3%. In cases of postnatal infection, PCRq detected 16 positive urines, the PCRq 12, and the cell culture 10. The urines showed viral loads ranging from 2,178 to 116,641 copies/ml. The genomic amplification technique PCRq in real time was more sensitive than the other techniques evaluated. This technique should be considered as a reference (gold standard), leaving the cell culture as a second diagnostic level. The low cost and the automation of PCRq would enable the screening for CMV infection in large neonatal and postnatal populations. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.

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Abdeldaim, Guma M K; Strålin, Kristoffer; Kirsebom, Leif A; Olcén, Per; Blomberg, Jonas; Herrmann, Björn

2009-08-01

A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissue.

Science.gov (United States)

Walton, Thomas J; Li, Geng; McCulloch, Thomas A; Seth, Rashmi; Powe, Desmond G; Bishop, Michael C; Rees, Robert C

2009-06-01

Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ERbeta) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann-Whitney U-test. Correlation coefficients were analyzed using Spearman's test. Significant positive correlations were seen when AR and AR-dependent PSA, and ERalpha and ERalpha-dependent PGR were compared, indicating a representative population of RNA transcripts. ERbeta gene expression was significantly over-expressed in the cancer group compared with benign controls (P cancer group (P prostate cancer specimens. In concert with recent studies the findings suggest differential production of ERbeta splice variants, which may play important roles in the genesis of prostate cancer. (c) 2009 Wiley-Liss, Inc.

Expression patterns of WRKY genes in di-haploid Populus simonii × P. nigra in response to salinity stress revealed by quantitative real-time PCR and RNA sequencing.

Science.gov (United States)

Wang, Shengji; Wang, Jiying; Yao, Wenjing; Zhou, Boru; Li, Renhua; Jiang, Tingbo

2014-10-01

Spatio-temporal expression patterns of 13 out of 119 poplar WRKY genes indicated dynamic and tissue-specific roles of WRKY family proteins in salinity stress tolerance. To understand the expression patterns of poplar WRKY genes under salinity stress, 51 of the 119 WRKY genes were selected from di-haploid Populus simonii × P. nigra by quantitative real-time PCR (qRT-PCR). We used qRT-PCR to profile the expression of the top 13 genes under salinity stress across seven time points, and employed RNA-Seq platforms to cross-validate it. Results demonstrated that all the 13 WRKY genes were expressed in root, stem, and leaf tissues, but their expression levels and overall patterns varied notably in these tissues. Regarding overall gene expression in roots, the 13 genes were significantly highly expressed at all six time points after the treatment, reaching the plateau of expression at hour 9. In leaves, the 13 genes were similarly up-regulated from 3 to 12 h in response to NaCl treatment. In stems, however, expression levels of the 13 genes did not show significant changes after the NaCl treatment. Regarding individual gene expression across the time points and the three tissues, the 13 genes can be classified into three clusters: the lowly expressed Cluster 1 containing PthWRKY28, 45 and 105; intermediately expressed Clusters 2 including PthWRKY56, 88 and 116; and highly expressed Cluster 3 consisting of PthWRKY41, 44, 51, 61, 62, 75 and 106. In general, genes in Cluster 2 and 3 displayed a dynamic pattern of "induced amplification-recovering", suggesting that these WRKY genes and corresponding pathways may play a critical role in mediating salt response and tolerance in a dynamic and tissue-specific manner.

Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results.

Directory of Open Access Journals (Sweden)

Andreas Garcia-Bardon

Full Text Available Real-time reverse transcription polymerase chain reaction (PCR is the gold standard for expression analysis. Designed to improve reproducibility and sensitivity, commercial kits are commonly used for the critical step of cDNA synthesis. The present study was designed to determine the impact of these kits. mRNA from mouse brains were pooled to create serial dilutions ranging from 0.0625 μg to 2 μg, which were transcribed into cDNA using four different commercial reverse-transcription kits. Next, we transcribed mRNA from brain tissue after acute brain injury and naïve mice into cDNA for qPCR. Depending on tested genes, some kits failed to show linear results in dilution series and revealed strong variations in cDNA yield. Absolute expression data in naïve and trauma settings varied substantially between these kits. Normalization with a housekeeping gene failed to reduce kit-dependent variations, whereas normalization eliminated differences when naïve samples from the same region were used. The study shows strong evidence that choice of commercial cDNA synthesis kit has a major impact on PCR results and, consequently, on comparability between studies. Additionally, it provides a solution to overcome this limitation by normalization with data from naïve samples. This simple step helps to compare mRNA expression data between different studies and groups.

Real-time imaging of radioisotope labeled compounds in a living plant

International Nuclear Information System (INIS)

Kanno, S.; Ohya, T.; Hayashi, Y.; Tanoi, K.; Nakanishi, T.M.

2007-01-01

We developed a quantitative, real-time imaging system of labeled compounds in a living plant. The system was composed of CsI scintillator to convert β-rays to visible light and an image intensifier unit (composed of GaAsP semiconductor and MCP; micro channel plate) to detect extremely weak light. When the sensitivity and resolution of the image of our system was compared with that of an imaging plate (IP), the sensitivity of our system (with 20 minutes) was higher than that of an IP, with similar quality to that of an IP. Using this system, the translocation of 32 P in a soybean plant tissue was shown in successive images. (author)

Detection of SYT-SSX mutant transcripts in formalin-fixed paraffin-embedded sarcoma tissues using one-step reverse transcriptase real-time PCR.

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Norlelawati, A T; Mohd Danial, G; Nora, H; Nadia, O; Zatur Rawihah, K; Nor Zamzila, A; Naznin, M

2016-04-01

Synovial sarcoma (SS) is a rare cancer and accounts for 5-10% of adult soft tissue sarcomas. Making an accurate diagnosis is difficult due to the overlapping histological features of SS with other types of sarcomas and the non-specific immunohistochemistry profile findings. Molecular testing is thus considered necessary to confirm the diagnosis since more than 90% of SS cases carry the transcript of t(X;18)(p11.2;q11.2). The purpose of this study is to diagnose SS at molecular level by testing for t(X;18) fusion-transcript expression through One-step reverse transcriptase real-time Polymerase Chain Reaction (PCR). Formalin-fixed paraffin-embedded tissue blocks of 23 cases of soft tissue sarcomas, which included 5 and 8 cases reported as SS as the primary diagnosis and differential diagnosis respectively, were retrieved from the Department of Pathology, Tengku Ampuan Afzan Hospital, Kuantan, Pahang. RNA was purified from the tissue block sections and then subjected to One-step reverse transcriptase real-time PCR using sequence specific hydrolysis probes for simultaneous detection of either SYT-SSX1 or SYT-SSX2 fusion transcript. Of the 23 cases, 4 cases were found to be positive for SYT-SSX fusion transcript in which 2 were diagnosed as SS whereas in the 2 other cases, SS was the differential diagnosis. Three cases were excluded due to failure of both amplification assays SYT-SSX and control β-2-microglobulin. The remaining 16 cases were negative for the fusion transcript. This study has shown that the application of One-Step reverse transcriptase real time PCR for the detection SYT-SSX transcript is feasible as an aid in confirming the diagnosis of synovial sarcoma.

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

Science.gov (United States)

Ogrean, Christy; Jackson, Ben; Covino, James

2010-01-01

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

Real-time systems

OpenAIRE

Badr, Salah M.; Bruztman, Donald P.; Nelson, Michael L.; Byrnes, Ronald Benton

1992-01-01

This paper presents an introduction to the basic issues involved in real-time systems. Both real-time operating sys and real-time programming languages are explored. Concurrent programming and process synchronization and communication are also discussed. The real-time requirements of the Naval Postgraduate School Autonomous Under Vehicle (AUV) are then examined. Autonomous underwater vehicle (AUV), hard real-time system, real-time operating system, real-time programming language, real-time sy...

Relative sensitivity of conventional and real-time PCR assays for detection of SFG Rickettsia in blood and tissue samples from laboratory animals.

Science.gov (United States)

Zemtsova, Galina E; Montgomery, Merrill; Levin, Michael L

2015-01-01

Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.

Enabling real-time ultrasound imaging of soft tissue mechanical properties by simplification of the shear wave motion equation.

Science.gov (United States)

Engel, Aaron J; Bashford, Gregory R

2015-08-01

Ultrasound based shear wave elastography (SWE) is a technique used for non-invasive characterization and imaging of soft tissue mechanical properties. Robust estimation of shear wave propagation speed is essential for imaging of soft tissue mechanical properties. In this study we propose to estimate shear wave speed by inversion of the first-order wave equation following directional filtering. This approach relies on estimation of first-order derivatives which allows for accurate estimations using smaller smoothing filters than when estimating second-order derivatives. The performance was compared to three current methods used to estimate shear wave propagation speed: direct inversion of the wave equation (DIWE), time-to-peak (TTP) and cross-correlation (CC). The shear wave speed of three homogeneous phantoms of different elastic moduli (gelatin by weight of 5%, 7%, and 9%) were measured with each method. The proposed method was shown to produce shear speed estimates comparable to the conventional methods (standard deviation of measurements being 0.13 m/s, 0.05 m/s, and 0.12 m/s), but with simpler processing and usually less time (by a factor of 1, 13, and 20 for DIWE, CC, and TTP respectively). The proposed method was able to produce a 2-D speed estimate from a single direction of wave propagation in about four seconds using an off-the-shelf PC, showing the feasibility of performing real-time or near real-time elasticity imaging with dedicated hardware.

Alchemy: A web 2.0 real-time quality assurance platform for human immunodeficiency Virus, hepatitis C Virus, and BK Virus quantitation assays

Directory of Open Access Journals (Sweden)

Emmanuel Agosto-Arroyo

2017-01-01

Full Text Available Background: The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT. Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs contain all the data elements necessary to do accurate quality assurance (QA reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Methods: Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV quantitation, hepatitis C virus (HCV quantitation, and BK virus (BKV quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Results: Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45–60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Conclusions: Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician

Alchemy: A Web 2.0 Real-time Quality Assurance Platform for Human Immunodeficiency Virus, Hepatitis C Virus, and BK Virus Quantitation Assays.

Science.gov (United States)

Agosto-Arroyo, Emmanuel; Coshatt, Gina M; Winokur, Thomas S; Harada, Shuko; Park, Seung L

2017-01-01

The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45-60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician-supervised resident/fellow programming projects as learning

Mathematical analysis of the real time array PCR (RTA PCR) process

NARCIS (Netherlands)

Dijksman, Johan Frederik; Pierik, A.

2012-01-01

Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the

A two-stage method of quantitative flood risk analysis for reservoir real-time operation using ensemble-based hydrologic forecasts

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Liu, P.

2013-12-01

Quantitative analysis of the risk for reservoir real-time operation is a hard task owing to the difficulty of accurate description of inflow uncertainties. The ensemble-based hydrologic forecasts directly depict the inflows not only the marginal distributions but also their persistence via scenarios. This motivates us to analyze the reservoir real-time operating risk with ensemble-based hydrologic forecasts as inputs. A method is developed by using the forecast horizon point to divide the future time into two stages, the forecast lead-time and the unpredicted time. The risk within the forecast lead-time is computed based on counting the failure number of forecast scenarios, and the risk in the unpredicted time is estimated using reservoir routing with the design floods and the reservoir water levels of forecast horizon point. As a result, a two-stage risk analysis method is set up to quantify the entire flood risks by defining the ratio of the number of scenarios that excessive the critical value to the total number of scenarios. The China's Three Gorges Reservoir (TGR) is selected as a case study, where the parameter and precipitation uncertainties are implemented to produce ensemble-based hydrologic forecasts. The Bayesian inference, Markov Chain Monte Carlo, is used to account for the parameter uncertainty. Two reservoir operation schemes, the real operated and scenario optimization, are evaluated for the flood risks and hydropower profits analysis. With the 2010 flood, it is found that the improvement of the hydrologic forecast accuracy is unnecessary to decrease the reservoir real-time operation risk, and most risks are from the forecast lead-time. It is therefore valuable to decrease the avarice of ensemble-based hydrologic forecasts with less bias for a reservoir operational purpose.

Real-time deep-tissue thermal sensing with sub-degree resolution by thermally improved Nd{sup 3+}:LaF{sub 3} multifunctional nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Rocha, Uéslen, E-mail: ueslen.silva@fis.ufal.br [Fluorescence Imaging Group, Departamento de Física de Materiales C-04, Instituto Nicolás Cabrera, Facultad de Ciencias, Universidad Autónoma de Madrid (Spain); Jacinto, Carlos; Kumar, Kagola Upendra [Grupo de Fotônica e Fluidos Complexos, Instituto de Física, Universidade Federal de Alagoas, 57072-970 Maceió, Alagoas (Brazil); López, Fernando J.; Bravo, David; Solé, José García [Fluorescence Imaging Group, Departamento de Física de Materiales C-04, Instituto Nicolás Cabrera, Facultad de Ciencias, Universidad Autónoma de Madrid (Spain); Jaque, Daniel, E-mail: daniel.jaque@uam.es [Fluorescence Imaging Group, Departamento de Física de Materiales C-04, Instituto Nicolás Cabrera, Facultad de Ciencias, Universidad Autónoma de Madrid (Spain); Instituto Ramón y Cajal de Investigación Sanitaria, Hospital Ramon y Cajal, Madrid 28034 (Spain)

2016-07-15

Nd{sup 3+} ion doped LaF{sub 3} dielectric nanoparticles have recently emerged as very attractive multifunctional nanoparticles capable of simultaneous sub-tissue heating and thermal sensing. Although they have been already used for selective photothermal treatment of cancer tumors in animal models, their real application as self-monitored photothermal agents require further optimization and development. Dynamic adjustment of the therapy parameters is mandatory for non-selective damage minimization. It would require real-time (sub-second) thermal sensing with a sub-degree thermal resolution. In this work we demonstrate that meeting this challenge is, indeed, possible by performing controlled thermal treatment on as-synthesized Nd{sup 3+} doped LaF{sub 3} nanoparticles. Temperature induced lattice ordering and defect re-combination have been concluded to induce, simultaneously, a line fluorescence narrowing, fluorescence brightness enhancement and a remarkable increment in thermal sensitivity. Ex-vivo experiments have demonstrated that, thanks to this multi-parameter optimization, Neodymium doped LaF{sub 3} nanoparticles are capable of real time sub-tissue thermal reading with a temperature resolution as low as 0.7 °C.

A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4.

Science.gov (United States)

Väisänen, E; Lahtinen, A; Eis-Hübinger, A M; Lappalainen, M; Hedman, K; Söderlund-Venermo, M

2014-01-01

Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes. Copyright © 2013 Elsevier B.V. All rights reserved.

Detection of Mycobacterium tuberculosis Complex in Paraffin-Embedded Tissues by the New Automated Abbott RealTime MTB Assay.

Science.gov (United States)

Fu, Yung-Chieh; Liao, I-Chuang; Chen, Hung-Mo; Yan, Jing-Jou

2016-07-01

The Abbott RealTime MTB assay, launched in June 2014, has been shown to have a competitive performance in the detection of the Mycobacterium tuberculosis (MTB) complex in respiratory specimens. The present study was conducted to investigate the usefulness of the Abbott MTB Realtime assay in the detection of MTB in formalin-fixed paraffin-embedded (FFPE) tissues. A total of 96 FFPE specimens obtained from microbiologically proven MTB cases (N=60) and nontuberculous Mycobacterium cases (N=36) were analyzed. The performance of the Abbott MTB Realtime assay was compared with that of the Roche Cobas TaqMan MTB assay. The overall sensitivity and specificity of the Abbott assay were 63.3% and 97.2%, respectively, compared with 11.7% and 100% for the Cobas assay. The detection rate of the Abbott assay was much higher among 37 acid-fast-positive specimens than among 23 acid-fast-negative specimens (89.3% versus 21.7%, respectively). The detection rate of the assay was higher among 29 resection specimens than among 31 small biopsy specimens (86.2% versus 41.9%, respectively). Our results suggest that the Abbott RealTime MTB assay can be used to differentiate MTB from nontuberculous mycobacterial infections in acid-fast-positive FFPE tissues. © 2016 by the Association of Clinical Scientists, Inc.

Clinical evaluation of a Mucorales-specific real-time PCR assay in tissue and serum samples.

Science.gov (United States)

Springer, Jan; Lackner, Michaela; Ensinger, Christian; Risslegger, Brigitte; Morton, Charles Oliver; Nachbaur, David; Lass-Flörl, Cornelia; Einsele, Hermann; Heinz, Werner J; Loeffler, Juergen

2016-12-01

Molecular diagnostic assays can accelerate the diagnosis of fungal infections and subsequently improve patient outcomes. In particular, the detection of infections due to Mucorales is still challenging for laboratories and physicians. The aim of this study was to evaluate a probe-based Mucorales-specific real-time PCR assay (Muc18S) using tissue and serum samples from patients suffering from invasive mucormycosis (IMM). This assay can detect a broad range of clinically relevant Mucorales species and can be used to complement existing diagnostic tests or to screen high-risk patients. An advantage of the Muc18S assay is that it exclusively detects Mucorales species allowing the diagnosis of Mucorales DNA without sequencing within a few hours. In paraffin-embedded tissue samples this PCR-based method allowed rapid identification of Mucorales in comparison with standard methods and showed 91 % sensitivity in the IMM tissue samples. We also evaluated serum samples, an easily accessible material, from patients at risk from IMM. Mucorales DNA was detected in all patients with probable/proven IMM (100 %) and in 29 % of the possible cases. Detection of IMM in serum could enable an earlier diagnosis (up to 21 days) than current methods including tissue samples, which were gained mainly post-mortem. A screening strategy for high-risk patients, which would enable targeted treatment to improve patient outcomes, is therefore possible.

Quantitative Real-time PCR detection of putrescine-producing Gram-negative bacteria

Directory of Open Access Journals (Sweden)

Kristýna Maršálková

2017-01-01

Full Text Available Biogenic amines are indispensable components of living cells; nevertheless these compounds could be toxic for human health in higher concentrations. Putrescine is supposed to be the major biogenic amine associated with microbial food spoilage. Development of reliable, fast and culture-independent molecular methods to detect bacteria producing biogenic amines deserves the attention, especially of the food industry in purpose to protect health. The objective of this study was to verify the newly designed primer sets for detection of two inducible genes adiA and speF together in Salmonella enterica and Escherichia coli genome by Real-time PCR. These forenamed genes encode enzymes in the metabolic pathway which leads to production of putrescine in Gram-negative bacteria. Moreover, relative expression of these genes was studied in E. coli CCM 3954 strain using Real-time PCR. In this study, sets of new primers for the detection two inducible genes (speF and adiA in Salmonella enterica and E. coli by Real-time PCR were designed and tested. Amplification efficiency of a Real-time PCR was calculated from the slope of the standard curves (adiA, speF, gapA. An efficiency in a range from 95 to 105 % for all tested reactions was achieved. The gene expression (R of adiA and speF genes in E. coli was varied depending on culture conditions. The highest gene expression of adiA and speF was observed at 6, 24 and 36 h (RadiA ~ 3, 5, 9; RspeF ~11, 10, 9; respectively after initiation of growth of this bacteria in nutrient broth medium enchired with amino acids. The results show that these primers could be used for relative quantification analysis of E. coli.

A triplex quantitative real-time PCR assay for differential detection of human adenovirus serotypes 2, 3 and 7.

Science.gov (United States)

Qiu, Fang-Zhou; Shen, Xin-Xin; Zhao, Meng-Chuan; Zhao, Li; Duan, Su-Xia; Chen, Chen; Qi, Ju-Ju; Li, Gui-Xia; Wang, Le; Feng, Zhi-Shan; Ma, Xue-Jun

2018-05-02

Human adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive. In the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated. The analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138). The proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7.

Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR.

Directory of Open Access Journals (Sweden)

Ruby Chandna

Full Text Available The real time quantitative reverse transcription PCR (qRT-PCR is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes, ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes, in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

Preliminary results of real-time in-vitro electronic speckle pattern interferometry (ESPI) measurements in otolaryngology

Science.gov (United States)

Conerty, Michelle D.; Castracane, James; Cacace, Anthony T.; Parnes, Steven M.; Gardner, Glendon M.; Miller, Mitchell B.

1995-05-01

Electronic Speckle Pattern Interferometry (ESPI) is a nondestructive optical evaluation technique that is capable of determining surface and subsurface integrity through the quantitative evaluation of static or vibratory motion. By utilizing state of the art developments in the areas of lasers, fiber optics and solid state detector technology, this technique has become applicable in medical research and diagnostics. Based on initial support from NIDCD and continued support from InterScience, Inc., we have been developing a range of instruments for improved diagnostic evaluation in otolaryngological applications based on the technique of ESPI. These compact fiber optic instruments are capable of making real time interferometric measurements of the target tissue. Ongoing development of image post- processing software is currently capable of extracting the desired quantitative results from the acquired interferometric images. The goal of the research is to develop a fully automated system in which the image processing and quantification will be performed in hardware in near real-time. Subsurface details of both the tympanic membrane and vocal cord dynamics could speed the diagnosis of otosclerosis, laryngeal tumors, and aid in the evaluation of surgical procedures.

Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed.

Science.gov (United States)

Robène, Isabelle; Perret, Marion; Jouen, Emmanuel; Escalon, Aline; Maillot, Marie-Véronique; Chabirand, Aude; Moreau, Aurélie; Laurent, Annie; Chiroleu, Frédéric; Pruvost, Olivier

2015-07-01

Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×10(3) CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae.

Science.gov (United States)

Takahashi, Hajime; Saito, Rumi; Miya, Satoko; Tanaka, Yuichiro; Miyamura, Natsumi; Kuda, Takashi; Kimura, Bon

2017-04-04

The family Enterobacteriaceae, members of which are widely distributed in the environment, includes many important human pathogens. In this study, a rapid real-time PCR method targeting rplP, coding for L16 protein, a component of the ribosome large subunit, was developed for enumerating Enterobacteriaceae strains, and its efficiency was evaluated using naturally contaminated food products. The rplP-targeted real-time PCR amplified Enterobacteriaceae species with Ct values of 14.0-22.8, whereas the Ct values for non-Enterobacteriaceae species were >30, indicating the specificity of this method for the Enterobacteriaceae. Using a calibration curve of Ct=-3.025 (log CFU/g)+37.35, which was calculated from individual plots of the cell numbers in different concentrations of 5 Enterobacteriaceae species, the rplP-targeted real-time PCR was applied to 51 food samples. A Enterobacteriaceae species in foods rapidly and accurately, and therefore, it can be used for the microbiological risk analysis of foods. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

Science.gov (United States)

Frølund, Maria; Björnelius, Eva; Lidbrink, Peter; Ahrens, Peter; Jensen, Jørgen Skov

2014-01-01

A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

Directory of Open Access Journals (Sweden)

Maria Frølund

Full Text Available A novel multiplex quantitative real-time polymerase chain reaction (qPCR for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae.

Directory of Open Access Journals (Sweden)

Meng Sun

Full Text Available The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA, elongation factor 1 (EF1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ribosomal protein S13 (RPS13, ribosomal protein S20 (RPS20, tubulin (TUB, and β-actin (ACTB were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1 were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands. 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults. 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C. To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83 was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae).

Science.gov (United States)

Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

2015-01-01

The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

An integrated real-time diagnostic concept using expert systems, qualitative reasoning and quantitative analysis

International Nuclear Information System (INIS)

Edwards, R.M.; Lee, K.Y.; Kumara, S.; Levine, S.H.

1989-01-01

An approach for an integrated real-time diagnostic system is being developed for inclusion as an integral part of a power plant automatic control system. In order to participate in control decisions and automatic closed loop operation, the diagnostic system must operate in real-time. Thus far, an expert system with real-time capabilities has been developed and installed on a subsystem at the Experimental Breeder Reactor (EBR-II) in Idaho, USA. Real-time simulation testing of advanced power plant concepts at the Pennsylvania State University has been developed and was used to support the expert system development and installation at EBR-II. Recently, the US National Science Foundation (NSF) and the US Department of Energy (DOE) have funded a Penn State research program to further enhance application of real-time diagnostic systems by pursuing implementation in a distributed power plant computer system including microprocessor based controllers. This paper summarizes past, current, planned, and possible future approaches to power plant diagnostic systems research at Penn State. 34 refs., 9 figs

Supersonic transient magnetic resonance elastography for quantitative assessment of tissue elasticity.

Science.gov (United States)

Liu, Yu; Liu, Jingfei; Fite, Brett Z; Foiret, Josquin; Ilovitsh, Asaf; Leach, J Kent; Dumont, Erik; Caskey, Charles F; Ferrara, Katherine W

2017-05-21

Non-invasive, quantitative methods to assess the properties of biological tissues are needed for many therapeutic and tissue engineering applications. Magnetic resonance elastography (MRE) has historically relied on external vibration to generate periodic shear waves. In order to focally assess a biomaterial or to monitor the response to ablative therapy, the interrogation of a specific region of interest by a focused beam is desirable and transient MRE (t-MRE) techniques have previously been developed to accomplish this goal. Also, strategies employing a series of discrete ultrasound pulses directed to increasing depths along a single line-of-sight have been designed to generate a quasi-planar shear wave. Such 'supersonic' excitations have been applied for ultrasound elasticity measurements. The resulting shear wave is higher in amplitude than that generated from a single excitation and the properties of the media are simply visualized and quantified due to the quasi-planar wave geometry and the opportunity to generate the wave at the site of interest. Here for the first time, we extend the application of supersonic methods by developing a protocol for supersonic transient magnetic resonance elastography (sst-MRE) using an MR-guided focused ultrasound system capable of therapeutic ablation. We apply the new protocol to quantify tissue elasticity in vitro using biologically-relevant inclusions and tissue-mimicking phantoms, compare the results with elasticity maps acquired with ultrasound shear wave elasticity imaging (US-SWEI), and validate both methods with mechanical testing. We found that a modified time-of-flight (TOF) method efficiently quantified shear modulus from sst-MRE data, and both the TOF and local inversion methods result in similar maps based on US-SWEI. With a three-pulse excitation, the proposed sst-MRE protocol was capable of visualizing quasi-planar shear waves propagating away from the excitation location and detecting differences in shear

Supersonic transient magnetic resonance elastography for quantitative assessment of tissue elasticity

Science.gov (United States)

Liu, Yu; Liu, Jingfei; Fite, Brett Z.; Foiret, Josquin; Ilovitsh, Asaf; Leach, J. Kent; Dumont, Erik; Caskey, Charles F.; Ferrara, Katherine W.

2017-05-01

Non-invasive, quantitative methods to assess the properties of biological tissues are needed for many therapeutic and tissue engineering applications. Magnetic resonance elastography (MRE) has historically relied on external vibration to generate periodic shear waves. In order to focally assess a biomaterial or to monitor the response to ablative therapy, the interrogation of a specific region of interest by a focused beam is desirable and transient MRE (t-MRE) techniques have previously been developed to accomplish this goal. Also, strategies employing a series of discrete ultrasound pulses directed to increasing depths along a single line-of-sight have been designed to generate a quasi-planar shear wave. Such ‘supersonic’ excitations have been applied for ultrasound elasticity measurements. The resulting shear wave is higher in amplitude than that generated from a single excitation and the properties of the media are simply visualized and quantified due to the quasi-planar wave geometry and the opportunity to generate the wave at the site of interest. Here for the first time, we extend the application of supersonic methods by developing a protocol for supersonic transient magnetic resonance elastography (sst-MRE) using an MR-guided focused ultrasound system capable of therapeutic ablation. We apply the new protocol to quantify tissue elasticity in vitro using biologically-relevant inclusions and tissue-mimicking phantoms, compare the results with elasticity maps acquired with ultrasound shear wave elasticity imaging (US-SWEI), and validate both methods with mechanical testing. We found that a modified time-of-flight (TOF) method efficiently quantified shear modulus from sst-MRE data, and both the TOF and local inversion methods result in similar maps based on US-SWEI. With a three-pulse excitation, the proposed sst-MRE protocol was capable of visualizing quasi-planar shear waves propagating away from the excitation location and detecting differences in shear

Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Larvae of Spodoptera litura Exposed to Azadirachtin Stress Conditions

OpenAIRE

Benshui Shu; Jingjing Zhang; Gaofeng Cui; Ranran Sun; Veeran Sethuraman; Xin Yi; Guohua Zhong

2018-01-01

Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this...

Relative sensitivity of conventional and real-time PCR assays for detection of SFG Rickettsia in blood and tissue samples from laboratory animals.

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Galina E Zemtsova

Full Text Available Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87. The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.

Sequential processing of quantitative phase images for the study of cell behaviour in real-time digital holographic microscopy.

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Zikmund, T; Kvasnica, L; Týč, M; Křížová, A; Colláková, J; Chmelík, R

2014-11-01

Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time-lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long-term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least-squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Real time magnetic resonance guided endomyocardial local delivery

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Corti, R; Badimon, J; Mizsei, G; Macaluso, F; Lee, M; Licato, P; Viles-Gonzalez, J F; Fuster, V; Sherman, W

2005-01-01

Objective: To investigate the feasibility of targeting various areas of left ventricle myocardium under real time magnetic resonance (MR) imaging with a customised injection catheter equipped with a miniaturised coil. Design: A needle injection catheter with a mounted resonant solenoid circuit (coil) at its tip was designed and constructed. A 1.5 T MR scanner with customised real time sequence combined with in-room scan running capabilities was used. With this system, various myocardial areas within the left ventricle were targeted and injected with a gadolinium-diethylenetriaminepentaacetic acid (DTPA) and Indian ink mixture. Results: Real time sequencing at 10 frames/s allowed clear visualisation of the moving catheter and its transit through the aorta into the ventricle, as well as targeting of all ventricle wall segments without further image enhancement techniques. All injections were visualised by real time MR imaging and verified by gross pathology. Conclusion: The tracking device allowed real time in vivo visualisation of catheters in the aorta and left ventricle as well as precise targeting of myocardial areas. The use of this real time catheter tracking may enable precise and adequate delivery of agents for tissue regeneration. PMID:15710717

Identification of circulating miRNA biomarkers based on global quantitative real-time PCR profiling

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Kang Kang

2012-02-01

Full Text Available Abstract MicroRNAs (miRNAs are small noncoding RNAs (18-25 nucleotides that regulate gene expression at the post-transcriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Deregulation of miRNAs in serum or plasma has been associated with many diseases including cancers and cardiovascular diseases, suggesting the possible use of miRNAs as diagnostic biomarkers. However, the detection of the small amount of miRNAs found in serum or plasma requires a method with high sensitivity and accuracy. Therefore, the current study describes polymerase chain reaction (PCR-based methods for measuring circulating miRNAs. Briefly, the procedure involves four major steps: (1 sample collection and preparation; (2 global miRNAs profiling using quantitative real-time PCR (qRT-PCR; (3 data normalization and analysis; and (4 selection and validation of miRNA biomarkers. In conclusion, qRT-PCR is a promising method for profiling of circulating miRNAs as biomarkers.

Identification of pyrG Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR Detection of Pleurotus ostreatus.

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Zheng, Shi; Shan, Luying; Zhuang, Yongliang; Shang, Ying

2018-03-01

As a well-known edible fungus rich in nutrients, Pleurotus ostreatus has been used as an alternative to expensive wild edible fungi. Specifically, the fact that using P. ostreatus instead of other expensive wild edible fungi has damaged the rights and interests of consumers. Among the existing methods for detection of food adulteration, the amplification of endogenous reference gene is the most accurate method. However, an ideal endogenous reference gene for P. ostreatus has yet to be developed. In this study, a DNA extraction method for P. ostreatus was optimized, and pyrG was selected as a species-specific gene through sequence alignment. This gene was subsequently subjected to qualitative and quantitative Polymerase Chain Reaction (PCR) assays with 3 different P. ostreatus varieties and 7 other species. A low detection limit of 5 pg/μL was obtained by TaqMan quantitative PCR, and no pyrG amplification product was observed in the 7 other species. No allelic variation was detected in P. ostreatus varieties. These experiments confirmed that pyrG was an ideal endogenous reference gene for the qualitative and real-time quantitative PCR detection of P. ostreatus. This method was also suitable for the examination of processed P. ostreatus samples and determination of adulteration in wild mushrooms. The pyrG gene was chosen as an ideal endogenous reference gene for the qualitative and real-time quantitative PCR detection of P. ostreatus, and the detection limit was 5 pg/μL for the quantification. This method is used not only for raw materials but also for processed P. ostreatus products and other processed mushroom foods. © 2018 Institute of Food Technologists®.

Quantitative Real Time PCR approach to study gene expression profile during prenatal growth of skeletal muscle in pig of Duroc and Pietrain breeds

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M. Cagnazzo

2010-01-01

Full Text Available The quantitative real time-PCR (QRT-PCR is a very sensitive method used to quantify mRNA level in gene expression analysis. Combining amplification, detection and quantification in a single step, allows a more accurate measurement compared to the traditional PCR end point analysis (Pfaffl, 2001; Bustin, 2002.

Assessing reference genes for accurate transcript normalization using quantitative real-time PCR in pearl millet [Pennisetum glaucum (L. R. Br].

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Prasenjit Saha

Full Text Available Pearl millet [Pennisetum glaucum (L. R.Br.], a close relative of Panicoideae food crops and bioenergy grasses, offers an ideal system to perform functional genomics studies related to C4 photosynthesis and abiotic stress tolerance. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR provides a sensitive platform to conduct such gene expression analyses. However, the lack of suitable internal control reference genes for accurate transcript normalization during qRT-PCR analysis in pearl millet is the major limitation. Here, we conducted a comprehensive assessment of 18 reference genes on 234 samples which included an array of different developmental tissues, hormone treatments and abiotic stress conditions from three genotypes to determine appropriate reference genes for accurate normalization of qRT-PCR data. Analyses of Ct values using Stability Index, BestKeeper, ΔCt, Normfinder, geNorm and RefFinder programs ranked PP2A, TIP41, UBC2, UBQ5 and ACT as the most reliable reference genes for accurate transcript normalization under different experimental conditions. Furthermore, we validated the specificity of these genes for precise quantification of relative gene expression and provided evidence that a combination of the best reference genes are required to obtain optimal expression patterns for both endogeneous genes as well as transgenes in pearl millet.

Identification of four squid species by quantitative real-time polymerase chain reaction.

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Ye, Jian; Feng, Junli; Liu, Shasha; Zhang, Yanping; Jiang, Xiaona; Dai, Zhiyuan

2016-02-01

Squids are distributed worldwide, including many species of commercial importance, and they are often made into varieties of flavor foods. The rapid identification methods for squid species especially their processed products, however, have not been well developed. In this study, quantitative real-time PCR (qPCR) systems based on specific primers and TaqMan probes have been established for rapid and accurate identification of four common squid species (Ommastrephes bartramii, Dosidicus gigas, Illex argentinus, Todarodes pacificus) in Chinese domestic market. After analyzing mitochondrial genes reported in GenBank, the mitochondrial cytochrome b (Cytb) gene was selected for O. bartramii detection, cytochrome c oxidase subunit I (COI) gene for D. gigas and T. Pacificus detection, ATPase subunit 6 (ATPase 6) gene for I. Argentinus detection, and 12S ribosomal RNA (12S rDNA) gene for designing Ommastrephidae-specific primers and probe. As a result, all the TaqMan systems are of good performance, and efficiency of each reaction was calculated by making standard curves. This method could detect target species either in single or mixed squid specimen, and it was applied to identify 12 squid processed products successfully. Thus, it would play an important role in fulfilling labeling regulations and squid fishery control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Real-time safety risk assessment based on a real-time location system for hydropower construction sites.

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Jiang, Hanchen; Lin, Peng; Fan, Qixiang; Qiang, Maoshan

2014-01-01

The concern for workers' safety in construction industry is reflected in many studies focusing on static safety risk identification and assessment. However, studies on real-time safety risk assessment aimed at reducing uncertainty and supporting quick response are rare. A method for real-time safety risk assessment (RTSRA) to implement a dynamic evaluation of worker safety states on construction site has been proposed in this paper. The method provides construction managers who are in charge of safety with more abundant information to reduce the uncertainty of the site. A quantitative calculation formula, integrating the influence of static and dynamic hazards and that of safety supervisors, is established to link the safety risk of workers with the locations of on-site assets. By employing the hidden Markov model (HMM), the RTSRA provides a mechanism for processing location data provided by the real-time location system (RTLS) and analyzing the probability distributions of different states in terms of false positives and negatives. Simulation analysis demonstrated the logic of the proposed method and how it works. Application case shows that the proposed RTSRA is both feasible and effective in managing construction project safety concerns.

Real-Time Video Stylization Using Object Flows.

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Lu, Cewu; Xiao, Yao; Tang, Chi-Keung

2017-05-05

We present a real-time video stylization system and demonstrate a variety of painterly styles rendered on real video inputs. The key technical contribution lies on the object flow, which is robust to inaccurate optical flow, unknown object transformation and partial occlusion as well. Since object flows relate regions of the same object across frames, shower-door effect can be effectively reduced where painterly strokes and textures are rendered on video objects. The construction of object flows is performed in real time and automatically after applying metric learning. To reduce temporal flickering, we extend the bilateral filtering into motion bilateral filtering. We propose quantitative metrics to measure the temporal coherence on structures and textures of our stylized videos, and perform extensive experiments to compare our stylized results with baseline systems and prior works specializing in watercolor and abstraction.

Accuracy of real-time shear wave elastography in the assessment of normal liver tissue in the guinea pig (cavia porcellus).

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Glińska-Suchocka, K; Kubiak, K; Spużak, J; Jankowski, M; Borusewicz, P

2017-03-28

Shear wave elastography is a novel technique enabling real-time measurement of the elasticity of liver tissue. The color map is superimposed on the classic ultrasound image of the assessed tissue, which enables a precise evaluation of the stiffness of the liver tissue. The aim of the study was to assess the stiffness of normal liver tissue in the guinea pig using shear wave elastography. The study was carried out on 36 guinea pigs using the SuperSonic Imagine Aixplorer scanner, and a 1 to 6 MH convex SC6-1 transducer. An ultrasound guided Try-Cut liver core needle biopsy was carried out in all the studied animals and the collected samples were examined to exclude pathological lesions. The mean liver tissue stiffness ranged from 0.89 to 5.40 kPa. We found that shear wave elastography is an easy, non-invasive technique that can be used to assess the stiffness of liver tissue. The obtained results can be used in future studies to assess the types and changes of liver tissue in the course of various types of liver disease.

Selection of Reliable Reference Genes for Gene Expression Studies in the Biofuel Plant Jatropha curcas Using Real-Time Quantitative PCR

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Lu Zhang

2013-12-01

Full Text Available Jatropha curcas is a promising renewable feedstock for biodiesel and bio-jet fuel production. To study gene expression in Jatropha in different tissues throughout development and under stress conditions, we examined a total of 11 typical candidate reference genes using real-time quantitative polymerase chain reaction (RT-qPCR analysis, which is widely used for validating transcript levels in gene expression studies. The expression stability of these candidate reference genes was assessed across a total of 20 samples, including various tissues at vegetative and reproductive stages and under desiccation and cold stress treatments. The results obtained using software qBasePLUS showed that the top-ranked reference genes differed across the sample subsets. The combination of actin, GAPDH, and EF1α would be appropriate as a reference panel for normalizing gene expression data across samples at different developmental stages; the combination of actin, GAPDH, and TUB5 should be used as a reference panel for normalizing gene expression data across samples under various abiotic stress treatments. With regard to different developmental stages, we recommend the use of actin and TUB8 for normalization at the vegetative stage and GAPDH and EF1α for normalization at the reproductive stage. For abiotic stress treatments, we recommend the use of TUB5 and TUB8 for normalization under desiccation stress and GAPDH and actin for normalization under cold stress. These results are valuable for future research on gene expression during development or under abiotic stress in Jatropha. To our knowledge, this is the first report on the stability of reference genes in Jatropha.

Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

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Lyng, Maria B; Laenkholm, Anne-Vibeke; Pallisgaard, Niels

2008-01-01

BACKGROUND: Quantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various...... means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent...... of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER...

A method for accurate detection of genomic microdeletions using real-time quantitative PCR

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Bassett Anne S

2005-12-01

Full Text Available Abstract Background Quantitative Polymerase Chain Reaction (qPCR is a well-established method for quantifying levels of gene expression, but has not been routinely applied to the detection of constitutional copy number alterations of human genomic DNA. Microdeletions or microduplications of the human genome are associated with a variety of genetic disorders. Although, clinical laboratories routinely use fluorescence in situ hybridization (FISH to identify such cryptic genomic alterations, there remains a significant number of individuals in which constitutional genomic imbalance is suspected, based on clinical parameters, but cannot be readily detected using current cytogenetic techniques. Results In this study, a novel application for real-time qPCR is presented that can be used to reproducibly detect chromosomal microdeletions and microduplications. This approach was applied to DNA from a series of patient samples and controls to validate genomic copy number alteration at cytoband 22q11. The study group comprised 12 patients with clinical symptoms of chromosome 22q11 deletion syndrome (22q11DS, 1 patient trisomic for 22q11 and 4 normal controls. 6 of the patients (group 1 had known hemizygous deletions, as detected by standard diagnostic FISH, whilst the remaining 6 patients (group 2 were classified as 22q11DS negative using the clinical FISH assay. Screening of the patients and controls with a set of 10 real time qPCR primers, spanning the 22q11.2-deleted region and flanking sequence, confirmed the FISH assay results for all patients with 100% concordance. Moreover, this qPCR enabled a refinement of the region of deletion at 22q11. Analysis of DNA from chromosome 22 trisomic sample demonstrated genomic duplication within 22q11. Conclusion In this paper we present a qPCR approach for the detection of chromosomal microdeletions and microduplications. The strategic use of in silico modelling for qPCR primer design to avoid regions of repetitive

Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR

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Wang, Chong; Robles, Francisco; Ramirez, Saul

2016-01-01

Gallibacterium is a genus within the family Pasteurellaceae characterized by a high level of phenotypic and genetic diversity. No diagnostic method has yet been described, which allows species-specific identification of Gallibacterium anatis. The aim of this study was to develop a real...... published conventional PCR method and culture-based identification, respectively. The detection rates were 97%, 78% and 34% for the current qPCR, the conventional PCR and the culture-based identification method, respectively. The qPCR assay was able to detect the gene gyrB in serial dilutions of 10......-time quantitative PCR (qPCR) method allowing species-specific identification and quantification of G. anatis. A G. anatis specific DNA sequence was identified in the gyrase subunit B gene (gyrB) and used to design a TaqMan probe and corresponding primers. The specificity of the assay was tested on 52 bacterial...

Real-Time PCR-Based Quantitation Method for the Genetically Modified Soybean Line GTS 40-3-2.

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Kitta, Kazumi; Takabatake, Reona; Mano, Junichi

2016-01-01

This chapter describes a real-time PCR-based method for quantitation of the relative amount of genetically modified (GM) soybean line GTS 40-3-2 [Roundup Ready(®) soybean (RRS)] contained in a batch. The method targets a taxon-specific soybean gene (lectin gene, Le1) and the specific DNA construct junction region between the Petunia hybrida chloroplast transit peptide sequence and the Agrobacterium 5-enolpyruvylshikimate-3-phosphate synthase gene (epsps) sequence present in GTS 40-3-2. The method employs plasmid pMulSL2 as a reference material in order to quantify the relative amount of GTS 40-3-2 in soybean samples using a conversion factor (Cf) equal to the ratio of the RRS-specific DNA to the taxon-specific DNA in representative genuine GTS 40-3-2 seeds.

Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification ▿

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Yankson, Kweku K.; Steck, Todd R.

2009-01-01

We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil. PMID:19633108

Quantitative analysis of myocardial tissue with digital autofluorescence microscopy

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Thomas Jensen

2016-01-01

Full Text Available Background: The opportunity offered by whole slide scanners of automated histological analysis implies an ever increasing importance of digital pathology. To go beyond the importance of conventional pathology, however, digital pathology may need a basic histological starting point similar to that of hematoxylin and eosin staining in conventional pathology. This study presents an automated fluorescence-based microscopy approach providing highly detailed morphological data from unstained microsections. This data may provide a basic histological starting point from which further digital analysis including staining may benefit. Methods: This study explores the inherent tissue fluorescence, also known as autofluorescence, as a mean to quantitate cardiac tissue components in histological microsections. Data acquisition using a commercially available whole slide scanner and an image-based quantitation algorithm are presented. Results: It is shown that the autofluorescence intensity of unstained microsections at two different wavelengths is a suitable starting point for automated digital analysis of myocytes, fibrous tissue, lipofuscin, and the extracellular compartment. The output of the method is absolute quantitation along with accurate outlines of above-mentioned components. The digital quantitations are verified by comparison to point grid quantitations performed on the microsections after Van Gieson staining. Conclusion: The presented method is amply described as a prestain multicomponent quantitation and outlining tool for histological sections of cardiac tissue. The main perspective is the opportunity for combination with digital analysis of stained microsections, for which the method may provide an accurate digital framework.

Real time automatic discriminating of ultrasonic flaws

International Nuclear Information System (INIS)

Suhairy Sani; Mohd Hanif Md Saad; Marzuki Mustafa; Mohd Redzwan Rosli

2009-01-01

This paper is concerned with the real time automatic discriminating of flaws from two categories; i. cracks (planar defect) and ii. Non-cracks (volumetric defect such as cluster porosity and slag) using pulse-echo ultrasound. The raw ultrasonic flaws signal were collected from a computerized robotic plane scanning system over the whole of each reflector as the primary source of data. The signal is then filtered and the analysis in both time and frequency domain were executed to obtain the selected feature. The real time feature analysis techniques measured the number of peaks, maximum index, pulse duration, rise time and fall time. The obtained features could be used to distinguish between quantitatively classified flaws by using various tools in artificial intelligence such as neural networks. The proposed algorithm and complete system were implemented in a computer software developed using Microsoft Visual BASIC 6.0 (author)

Determination of the Elasticity of Breast Tissue during the Menstrual Cycle Using Real-Time Shear Wave Elastography.

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Li, Xiang; Wang, Jian-Nan; Fan, Zhi-Ying; Kang, Shu; Liu, Yan-Jun; Zhang, Yi-Xia; Wang, Xue-Mei

2015-12-01

We examined breast tissue elasticity during the menstrual cycle using real-time shear wave elastography (RT-SWE), a recent technique developed for soft tissue imaging. Written informed consent for RT-SWE was obtained from all eligible patients, who were healthy women aged between 19 and 52 y. Young's moduli of the breast tissue in the early follicular, late phase and luteal phase were compared. There were no significant differences in the mean, maximum and minimum elasticity values (Emean, Emax and Emin) and standard deviation (ESD). RT-SWE of glandular tissue revealed that ESD was increased in the early follicular phase compared with the luteal phase. Means ± SD of Emin, Emax and Emean in glandular tissue were 5.174 ± 2.138, 8.308 ± 3.166 and 6.593 ± 2.510, respectively, and in adipose tissue, 3.589 ± 2.083, 6.733 ± 3.522 and 4.857 ± 2.564, respectively. There were no significant differences in stiffness between glandular and adipose tissues throughout the menstrual cycle, but glandular tissue stiffness was lower in the luteal phase than in the early follicular phase. On the basis of these observations in normal healthy women, we believe we have obtained sufficient information to establish the baseline changes in human breast elasticity during the menstrual cycle. In the future, we intend to compare the elasticity values of healthy breast tissue with those of breast tissue affected by various pathologies. Our results reveal the significant potential of RT-SWE in the rapid and non-invasive clinical diagnosis of breast diseases, such as breast cancers. Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Detection of Histoplasma capsulatum from clinical specimens by cycling probe-based real-time PCR and nested real-time PCR.

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Muraosa, Yasunori; Toyotome, Takahito; Yahiro, Maki; Watanabe, Akira; Shikanai-Yasuda, Maria Aparecida; Kamei, Katsuhiko

2016-05-01

We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Selection and validation of appropriate reference genes for quantitative real-time PCR analysis in Salvia hispanica.

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Rahul Gopalam

Full Text Available Quantitative real-time polymerase chain reaction (qRT-PCR has become the most popular choice for gene expression studies. For accurate expression analysis, it is pertinent to select a stable reference gene to normalize the data. It is now known that the expression of internal reference genes varies considerably during developmental stages and under different experimental conditions. For Salvia hispanica, an economically important oilseed crop, there are no reports of stable reference genes till date. In this study, we chose 13 candidate reference genes viz. Actin11 (ACT, Elongation factor 1-alpha (EF1-α, Eukaryotic translation initiation factor 3E (ETIF3E, alpha tubulin (α-TUB, beta tubulin (β-TUB, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cyclophilin (CYP, Clathrin adaptor complex (CAC, Serine/threonine-protein phosphatase 2A (PP2A, FtsH protease (FtsH, 18S ribosomal RNA (18S rRNA, S-adenosyl methionine decarboxylase (SAMDC and Rubisco activase (RCA and the expression levels of these genes were assessed in a diverse set of tissue samples representing vegetative stages, reproductive stages and various abiotic stress treatments. Two of the widely used softwares, geNorm and Normfinder were used to evaluate the expression stabilities of these 13 candidate reference genes under different conditions. Results showed that GAPDH and CYP expression remain stable throughout in the different abiotic stress treatments, CAC and PP2A expression were relatively stable under reproductive stages and α-TUB, PP2A and ETIF3E were found to be stably expressed in vegetative stages. Further, the expression levels of Diacylglycerol acyltransferase (DGAT1, a key enzyme in triacylglycerol synthesis was analyzed to confirm the validity of reference genes identified in the study. This is the first systematic study of selection of reference genes in S. hispanica, and will benefit future expression studies in this crop.

Real Time Revisited

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Allen, Phillip G.

1985-12-01

The call for abolishing photo reconnaissance in favor of real time is once more being heard. Ten years ago the same cries were being heard with the introduction of the Charge Coupled Device (CCD). The real time system problems that existed then and stopped real time proliferation have not been solved. The lack of an organized program by either DoD or industry has hampered any efforts to solve the problems, and as such, very little has happened in real time in the last ten years. Real time is not a replacement for photo, just as photo is not a replacement for infra-red or radar. Operational real time sensors can be designed only after their role has been defined and improvements made to the weak links in the system. Plodding ahead on a real time reconnaissance suite without benefit of evaluation of utility will allow this same paper to be used ten years from now.

Near-real-time materials accountancy in an international perspective or will the real near-real-time materials accountancy please stand up

International Nuclear Information System (INIS)

Lovett, J.E.

1981-01-01

During the 1970's the IAEA gave considerable attention to the question of what its quantitative goals should be. These discussions led, in January 1980, to a set of provisional guidelines which considered both abrupt and protracted diversion possibilities. In a search for an effective means of achieving these goals at large bulk processing facilities, two technological concepts have been put forward. One concept, commonly referred to as near-real-time materials accountancy, is reviewed in this paper. 9 refs

Comparison of high and low virulence serotypes of Actinobacillus pleuropneumoniae by quantitative real-time PCR

DEFF Research Database (Denmark)

Schou, Kirstine Klitgaard; Angen, Øystein; Boye, Mette

PCR data. Preliminary results showed that in both serotype 2 and serotype 6, the toxin producing gene apxIV was the most highly expressed of the investigated genes. The major difference observed between the two serotypes was that apfA, involved in type IV vili production, was significantly upregulated...... of high virulence while serotype 6 strains are normally found to be less pathogenic. To gain an understanding of the differential virulence of serotype 2 and 6, the expression of a panel of Ap genes during infection of porcine epithelial lung cells (SJPL) were examined by quantitative real-time PCR (q...... to be important for early establishment of the bacteria in the host were examined by qPCR. The genes examined were apfA, coding for a subunit of Type IV pili, kdsB coding for a gene involved in lippopolysacceride biosynthesis, and pgaB which is involved in biofilm formation, all three believed to be important...

Validation of reference genes for quantitative expression analysis by real-time rt-PCR in four lepidopteran insects.

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Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei

2012-01-01

Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae), Chilo suppressalis Walker (Crambidae), and Spodoptera exigua Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ΔCt analysis were used to evaluate these HKGs. Across different developmental stages, actin A1 was the most stable in P. xylostella and C. suppressalis, but it was the least stable in B. mori and S. exigua. Rp49 and GAPDH were the most stable in B. mori and S. exigua, respectively. In different tissues, GAPDH, E2F, and Rp49 were the most stable in B. mori, S. exigua, and C. suppressalis, respectively. The relative abundances of Siwi genes estimated by 2(-ΔΔCt) method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR.

Real-time patient-specific finite element analysis of internal stresses in the soft tissues of a residual limb: a new tool for prosthetic fitting.

Science.gov (United States)

Portnoy, S; Yarnitzky, G; Yizhar, Z; Kristal, A; Oppenheim, U; Siev-Ner, I; Gefen, A

2007-01-01

Fitting of a prosthetic socket is a critical stage in the process of rehabilitation of a trans-tibial amputation (TTA) patient, since a misfit may cause pressure ulcers or a deep tissue injury (DTI: necrosis of the muscle flap under intact skin) in the residual limb. To date, prosthetic fitting typically depends on the subjective skills of the prosthetist, and is not supported by biomedical instrumentation that allows evaluation of the quality of fitting. Specifically, no technology is presently available to provide real-time continuous information on the internal distribution of mechanical stresses in the residual limb during fitting of the prosthesis, or while using it and this severely limits patient evaluations. In this study, a simplified yet clinically oriented patient-specific finite element (FE) model of the residual limb was developed for real-time stress analysis. For this purpose we employed a custom-made FE code that continuously calculates internal stresses in the residual limb, based on boundary conditions acquired in real-time from force sensors, located at the limb-prosthesis interface. Validation of the modeling system was accomplished by means of a synthetic phantom of the residual limb, which allowed simultaneous measurements of interface pressures and internal stresses. Human studies were conducted subsequently in five TTA patients. The dimensions of bones and soft tissues were obtained from X-rays of the residual limb of each patient. An indentation test was performed in order to obtain the effective elastic modulus of the soft tissues of the residual limb. Seven force sensors were placed between the residual limb and the prosthetic liner, and subjects walked on a treadmill during analysis. Generally, stresses under the shinbones were approximately threefold higher than stresses at the soft tissues behind the bones. Usage of a thigh corset decreased the stresses in the residual limb during gait by approximately 80%. Also, the stresses

A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl

Science.gov (United States)

Smith, Matthew M.; Schmutz, Joel A.; Apelgren, Chloe; Ramey, Andy M.

2015-01-01

Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n = 105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R2 = 0.694, P = 0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species.

Real time analysis of brain tissue by direct combination of ultrasonic surgical aspiration and sonic spray mass spectrometry.

Science.gov (United States)

Schäfer, Karl-Christian; Balog, Júlia; Szaniszló, Tamás; Szalay, Dániel; Mezey, Géza; Dénes, Júlia; Bognár, László; Oertel, Matthias; Takáts, Zoltán

2011-10-15

Direct combination of cavitron ultrasonic surgical aspirator (CUSA) and sonic spray ionization mass spectrometry is presented. A commercially available ultrasonic surgical device was coupled to a Venturi easy ambient sonic-spray ionization (V-EASI) source by directly introducing liquified tissue debris into the Venturi air jet pump. The Venturi air jet pump was found to efficiently nebulize the suspended tissue material for gas phase ion production. The ionization mechanism involving solely pneumatic spraying was associated with that of sonic spray ionization. Positive and negative ionization spectra were obtained from brain and liver samples reflecting the primary application areas of the surgical device. Mass spectra were found to feature predominantly complex lipid-type constituents of tissues in both ion polarity modes. Multiply charged peptide anions were also detected. The influence of instrumental settings was characterized in detail. Venturi pump geometry and flow parameters were found to be critically important in ionization efficiency. Standard solutions of phospholipids and peptides were analyzed in order to test the dynamic range, sensitivity, and suppression effects. The spectra of the intact tissue specimens were found to be highly specific to the histological tissue type. The principal component analysis (PCA) and linear discriminant analysis (LDA) based data analysis method was developed for real-time tissue identification in a surgical environment. The method has been successfully tested on post-mortem and ex vivo human samples including astrocytomas, meningeomas, metastatic brain tumors, and healthy brain tissue. © 2011 American Chemical Society

Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: applications and considerations.

Science.gov (United States)

Kim, Jaai; Lim, Juntaek; Lee, Changsoo

2013-12-01

Quantitative real-time PCR (qPCR) has been widely used in recent environmental microbial ecology studies as a tool for detecting and quantifying microorganisms of interest, which aids in better understandings of the complexity of wastewater microbial communities. Although qPCR can be used to provide more specific and accurate quantification than other molecular techniques, it does have limitations that must be considered when applying it in practice. This article reviews the principle of qPCR quantification and its applications to microbial ecology studies in various wastewater treatment environments. Here we also address several limitations of qPCR-based approaches that can affect the validity of quantification data: template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database. Even with such limitations, qPCR is reportedly among the best methods for quantitatively investigating environmental microbial communities. The application of qPCR is and will continue to be increasingly common in studies of wastewater treatment systems. To obtain reliable analyses, however, the limitations that have often been overlooked must be carefully considered when interpreting the results. Copyright © 2013 Elsevier Inc. All rights reserved.

Real-time intravital imaging of pH variation associated with osteoclast activity.

Science.gov (United States)

Maeda, Hiroki; Kowada, Toshiyuki; Kikuta, Junichi; Furuya, Masayuki; Shirazaki, Mai; Mizukami, Shin; Ishii, Masaru; Kikuchi, Kazuya

2016-08-01

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

Instruments for radiation measurement in life sciences (5). Development of imaging technology in life science. 4. Real-time bioradiography

International Nuclear Information System (INIS)

Sasaki, Toru; Iwamoto, Akinori; Tsuboi, Hisashi; Katoh, Toru; Kudo, Hiroyuki; Kazawa, Erito; Watanabe, Yasuyoshi

2006-01-01

Real-time bioradiography, new bioradiography method, can collect and produce image of metabolism and function of cell in real-time. The principles of instrumentation, development process and the application examples of neuroscience and biomedical gerontology are stated. The bioradiography method, the gas-tissue live-cell autoradiography method and the real-time bioradiography method are explained. As the application examples, the molecular mechanism of oxidative stress at brain ischemia and the analysis of SOD gene knockout animals are reported. Comparison between FDG-PET of epileptic brain and FDG- bioradiography image of live-cell of brain tissue, the real-time bioradiography system, improvement of image by surface treatment, the detection limit of β + ray from F 18 , image of living-slices of brain tissue by FDG-real-time bioradiography and radioluminography, continuous FDG image of living-slices of rat brain tissue, and analysis of carbohydrate metabolism of living-slices of brain tissue of mouse lacking SOD gene during aerophobia and reoxygenation process are reported. (S.Y.)

Multifactorial Optimization of Contrast-Enhanced Nanofocus Computed Tomography for Quantitative Analysis of Neo-Tissue Formation in Tissue Engineering Constructs.

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Maarten Sonnaert

Full Text Available To progress the fields of tissue engineering (TE and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells/tissue combined with scaffolds becomes essential. In this study, we have defined the most optimal staining conditions for contrast-enhanced nanofocus computed tomography for three dimensional visualization and quantitative analysis of in vitro engineered neo-tissue (i.e. extracellular matrix containing cells in perfusion bioreactor-developed Ti6Al4V constructs. A fractional factorial 'design of experiments' approach was used to elucidate the influence of the staining time and concentration of two contrast agents (Hexabrix and phosphotungstic acid and the neo-tissue volume on the image contrast and dataset quality. Additionally, the neo-tissue shrinkage that was induced by phosphotungstic acid staining was quantified to determine the operating window within which this contrast agent can be accurately applied. For Hexabrix the staining concentration was the main parameter influencing image contrast and dataset quality. Using phosphotungstic acid the staining concentration had a significant influence on the image contrast while both staining concentration and neo-tissue volume had an influence on the dataset quality. The use of high concentrations of phosphotungstic acid did however introduce significant shrinkage of the neo-tissue indicating that, despite sub-optimal image contrast, low concentrations of this staining agent should be used to enable quantitative analysis. To conclude, design of experiments allowed us to define the most optimal staining conditions for contrast-enhanced nanofocus computed tomography to be used as a routine screening tool of neo-tissue formation in Ti6Al4V constructs, transforming it into a robust three dimensional quality control methodology.

A quantitative real time polymerase chain reaction approach for estimating processed animal proteins in feed: preliminary data

Directory of Open Access Journals (Sweden)

Maria Cesarina Abete

2013-04-01

Full Text Available Lifting of the ban on the use of processed animal proteins (PAPs from non-ruminants in non-ruminant feed is in the wind, avoiding intraspecies recycling. Discrimination of species will be performed through polymerase chain reaction (PCR, which is at a moment a merely qualitative method. Nevertheless, quantification of PAPs in feed is needed. The aim of this study was to approach the quantitative determination of PAPs in feed through Real Time (RT-PCR technique; three different protocols picked up from the literature were tested. Three different kind of matrices were examined: pure animal meals (bovine, chicken and pork; one feed sample certified by the European reference laboratory on animal proteins (EURL AP in feed spiked with 0.1% bovine meal; and genomic DNAs from bovine, chicken and pork muscles. The limit of detection (LOD of the three protocols was set up. All the results obtained from the three protocols considered failed in the quantification process, most likely due to the uncertain copy numbers of the analytical targets chosen. This preliminary study will allow us to address further investigations, with the purpose of developing a RT-PCR quantitative method.

Development of a real-time and quantitative thrombus sensor for an extracorporeal centrifugal blood pump by near-infrared light.

Science.gov (United States)

Sakota, Daisuke; Fujiwara, Tatsuki; Ohuchi, Katsuhiro; Kuwana, Katsuyuki; Yamazaki, Hiroyuki; Kosaka, Ryo; Nishida, Masahiro; Mizuno, Tomohiro; Arai, Hirokuni; Maruyama, Osamu

2018-01-01

We developed an optical thrombus sensor for a monopivot extracorporeal centrifugal blood pump. In this study, we investigated its quantitative performance for thrombus detection in acute animal experiments of left ventricular assist using the pump on pathogen-free pigs. Optical fibers were set in the driver unit of the pump. The incident light at the near-infrared wavelength of 810 nm was aimed at the pivot bearing, and the resulting scattered light was guided to the optical fibers. The detected signal was analyzed to obtain the thrombus formation level. As a result, real-time and quantitative monitoring of the thrombus surface area on the pivot bearing was achieved with an accuracy of 3.6 ± 2.3 mm 2 . In addition, the sensing method using the near-infrared light was not influenced by changes in the oxygen saturation and the hematocrit. It is expected that the developed sensor will be useful for optimal anticoagulation management for long-term extracorporeal circulation therapies.

Virtual reality myringotomy simulation with real-time deformation: development and validity testing.

Science.gov (United States)

Ho, Andrew K; Alsaffar, Hussain; Doyle, Philip C; Ladak, Hanif M; Agrawal, Sumit K

2012-08-01

Surgical simulation is becoming an increasingly common training tool in residency programs. The first objective was to implement real-time soft-tissue deformation and cutting into a virtual reality myringotomy simulator. The second objective was to test the various implemented incision algorithms to determine which most accurately represents the tympanic membrane during myringotomy. Descriptive and face-validity testing. A deformable tympanic membrane was developed, and three soft-tissue cutting algorithms were successfully implemented into the virtual reality myringotomy simulator. The algorithms included element removal, direction prediction, and Delaunay cutting. The simulator was stable and capable of running in real time on inexpensive hardware. A face-validity study was then carried out using a validated questionnaire given to eight otolaryngologists and four senior otolaryngology residents. Each participant was given an adaptation period on the simulator, was blinded to the algorithm being used, and was presented the three algorithms in a randomized order. A virtual reality myringotomy simulator with real-time soft-tissue deformation and cutting was successfully developed. The simulator was stable, ran in real time on inexpensive hardware, and incorporated haptic feedback and stereoscopic vision. The Delaunay cutting algorithm was found to be the most realistic algorithm representing the incision during myringotomy (P virtual reality myringotomy simulator is being developed and now integrates a real-time deformable tympanic membrane that appears to have face validity. Further development and validation studies are necessary before the simulator can be studied with respect to training efficacy and clinical impact. Copyright © 2012 The American Laryngological, Rhinological, and Otological Society, Inc.

Quantitative EEG analysis using error reduction ratio-causality test; validation on simulated and real EEG data.

Science.gov (United States)

Sarrigiannis, Ptolemaios G; Zhao, Yifan; Wei, Hua-Liang; Billings, Stephen A; Fotheringham, Jayne; Hadjivassiliou, Marios

2014-01-01

To introduce a new method of quantitative EEG analysis in the time domain, the error reduction ratio (ERR)-causality test. To compare performance against cross-correlation and coherence with phase measures. A simulation example was used as a gold standard to assess the performance of ERR-causality, against cross-correlation and coherence. The methods were then applied to real EEG data. Analysis of both simulated and real EEG data demonstrates that ERR-causality successfully detects dynamically evolving changes between two signals, with very high time resolution, dependent on the sampling rate of the data. Our method can properly detect both linear and non-linear effects, encountered during analysis of focal and generalised seizures. We introduce a new quantitative EEG method of analysis. It detects real time levels of synchronisation in the linear and non-linear domains. It computes directionality of information flow with corresponding time lags. This novel dynamic real time EEG signal analysis unveils hidden neural network interactions with a very high time resolution. These interactions cannot be adequately resolved by the traditional methods of coherence and cross-correlation, which provide limited results in the presence of non-linear effects and lack fidelity for changes appearing over small periods of time. Copyright © 2013 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

Real-Time Imaging System for the OpenPET

Science.gov (United States)

Tashima, Hideaki; Yoshida, Eiji; Kinouchi, Shoko; Nishikido, Fumihiko; Inadama, Naoko; Murayama, Hideo; Suga, Mikio; Haneishi, Hideaki; Yamaya, Taiga

2012-02-01

The OpenPET and its real-time imaging capability have great potential for real-time tumor tracking in medical procedures such as biopsy and radiation therapy. For the real-time imaging system, we intend to use the one-pass list-mode dynamic row-action maximum likelihood algorithm (DRAMA) and implement it using general-purpose computing on graphics processing units (GPGPU) techniques. However, it is difficult to make consistent reconstructions in real-time because the amount of list-mode data acquired in PET scans may be large depending on the level of radioactivity, and the reconstruction speed depends on the amount of the list-mode data. In this study, we developed a system to control the data used in the reconstruction step while retaining quantitative performance. In the proposed system, the data transfer control system limits the event counts to be used in the reconstruction step according to the reconstruction speed, and the reconstructed images are properly intensified by using the ratio of the used counts to the total counts. We implemented the system on a small OpenPET prototype system and evaluated the performance in terms of the real-time tracking ability by displaying reconstructed images in which the intensity was compensated. The intensity of the displayed images correlated properly with the original count rate and a frame rate of 2 frames per second was achieved with average delay time of 2.1 s.

Brain physiological state evaluated by real-time multiparametric tissue spectroscopy in vivo

Science.gov (United States)

Mayevsky, Avraham; Barbiro-Michaely, Efrat; Kutai-Asis, Hofit; Deutsch, Assaf; Jaronkin, Alex

2004-07-01

The significance of normal mitochondrial function in cellular energy homeostasis as well as its involvement in acute and chronic neurodegenerative disease was reviewed recently (Nicholls & Budd. Physiol Rev. 80: 315-360, 2000). Nevertheless, monitoring of mitochondrial function in vivo and real time mode was not used by many investigators and is very rare in clinical practice. The main principle tool available for the evaluation of mitochondrial function is the monitoring of NADH fluorescence. In order to interpret correctly the changes in NADH redox state in vivo, it is necessary to correlate this signal to other parameters, reflecting O2 supply to the brain. Therefore, we have developed and applied a multiparametric optical monitoring system, by which microcirculatory blood flow and hemoglobin oxygenation is measured, together with mitochondrial NADH fluorescence. Since the calibration of these signals is not in absolute units, the simultaneous monitoring provide a practical tool for the interpretation of brain functional state under various pathophysiological conditions. The monitoring system combines a time-sharing fluorometer-reflectometer for the measurement of NADH fluorescence and hemoglobin oxygenation as well as a laser Doppler flowmeter for the recording of microcirculatory blood flow. A combined fiber optic probe was located on the surface of the brain using a skull cemented cannula. Rats and gerbils were exposed to anoxia, ischemia and spreading depression and the functional state of the brain was evaluated. The results showed a clear correlation between O2 supply/demand as well as, energy balance under the various pathophysiological conditions. This monitoring approach could be adapted to clinical monitoring of tissue vitality.

Real-time imaging of quantum entanglement.

Science.gov (United States)

Fickler, Robert; Krenn, Mario; Lapkiewicz, Radek; Ramelow, Sven; Zeilinger, Anton

2013-01-01

Quantum Entanglement is widely regarded as one of the most prominent features of quantum mechanics and quantum information science. Although, photonic entanglement is routinely studied in many experiments nowadays, its signature has been out of the grasp for real-time imaging. Here we show that modern technology, namely triggered intensified charge coupled device (ICCD) cameras are fast and sensitive enough to image in real-time the effect of the measurement of one photon on its entangled partner. To quantitatively verify the non-classicality of the measurements we determine the detected photon number and error margin from the registered intensity image within a certain region. Additionally, the use of the ICCD camera allows us to demonstrate the high flexibility of the setup in creating any desired spatial-mode entanglement, which suggests as well that visual imaging in quantum optics not only provides a better intuitive understanding of entanglement but will improve applications of quantum science.

Transrectal real-time tissue elastography targeted biopsy coupled with peak strain index improves the detection of clinically important prostate cancer.

Science.gov (United States)

Ma, Qi; Yang, Dong-Rong; Xue, Bo-Xin; Wang, Cheng; Chen, Han-Bin; Dong, Yun; Wang, Cai-Shan; Shan, Yu-Xi

2017-07-01

The focus of the present study was to evaluate transrectal real-time tissue elastography (RTE)-targeted two-core biopsy coupled with peak strain index for the detection of prostate cancer (PCa) and to compare this method with 10-core systematic biopsy. A total of 141 patients were enrolled for evaluation. The diagnostic value of peak strain index was assessed using a receiver operating characteristic curve. The cancer detection rates of the two approaches and corresponding positive cores and Gleason score were compared. The cancer detection rate per core in the RTE-targeted biopsy (44%) was higher compared with that in systematic biopsy (30%). The peak strain index value of PCa was higher compared with that of the benign lesion. PCa was detected with the highest sensitivity (87.5%) and specificity (85.5%) using the threshold value of a peak strain index of ≥5.97 with an area under the curve value of 0.95. When the Gleason score was ≥7, RTE-targeted biopsy coupled with peak strain index detected 95.6% of PCa cases, but 84.4% were detected using systematic biopsy. Peak strain index as a quantitative parameter may improve the differentiation of PCa from benign lesions in the prostate peripheral zone. Transrectal RTE-targeted biopsy coupled with peak strain index may enhance the detection of clinically significant PCa, particularly when combined with systematic biopsy.

Evaluation of changes in periodontal bacteria in healthy dogs over 6 months using quantitative real-time PCR.

Science.gov (United States)

Maruyama, N; Mori, A; Shono, S; Oda, H; Sako, T

2018-03-01

Porphyromonas gulae, Tannerella forsythia and Campylobacter rectus are considered dominant periodontal pathogens in dogs. Recently, quantitative real-time PCR (qRT-PCR) methods have been used for absolute quantitative determination of oral bacterial counts. The purpose of the present study was to establish a standardized qRT-PCR procedure to quantify bacterial counts of the three target periodontal bacteria (P. gulae, T. forsythia and C. rectus). Copy numbers of the three target periodontal bacteria were evaluated in 26 healthy dogs. Then, changes in bacterial counts of the three target periodontal bacteria were evaluated for 24 weeks in 7 healthy dogs after periodontal scaling. Analytical evaluation of each self-designed primer indicated acceptable analytical imprecision. All 26 healthy dogs were found to be positive for P. gulae, T. forsythia and C. rectus. Median total bacterial counts (copies/ng) of each target genes were 385.612 for P. gulae, 25.109 for T. forsythia and 5.771 for C. rectus. Significant differences were observed between the copy numbers of the three target periodontal bacteria. Periodontal scaling reduced median copy numbers of the three target periodontal bacteria in 7 healthy dogs. However, after periodontal scaling, copy numbers of all three periodontal bacteria significantly increased over time (pperiodontal bacteria in dogs. Furthermore, the present study has revealed that qRT-PCR method can be considered as a new objective evaluation system for canine periodontal disease. Copyright© by the Polish Academy of Sciences.

Improved quantification accuracy for duplex real-time PCR detection of genetically modified soybean and maize in heat processed foods

Directory of Open Access Journals (Sweden)

CHENG Fang

2013-04-01

Full Text Available Real-time PCR technique has been widely used in quantitative GMO detection in recent years.The accuracy of GMOs quantification based on the real-time PCR methods is still a difficult problem,especially for the quantification of high processed samples.To develop the suitable and accurate real-time PCR system for high processed GM samples,we made ameliorations to several real-time PCR parameters,including re-designed shorter target DNA fragment,similar lengths of amplified endogenous and exogenous gene targets,similar GC contents and melting temperatures of PCR primers and TaqMan probes.Also,one Heat-Treatment Processing Model (HTPM was established using soybean flour samples containing GM soybean GTS 40-3-2 to validate the effectiveness of the improved real-time PCR system.Tested results showed that the quantitative bias of GM content in heat processed samples were lowered using the new PCR system.The improved duplex real-time PCR was further validated using processed foods derived from GM soybean,and more accurate GM content values in these foods was also achieved.These results demonstrated that the improved duplex real-time PCR would be quite suitable in quantitative detection of high processed food products.

A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

Science.gov (United States)

Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

2011-12-01

Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Real-time in vivo imaging of butterfly wing development: revealing the cellular dynamics of the pupal wing tissue.

Directory of Open Access Journals (Sweden)

Masaki Iwata

Full Text Available Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the

Real-time in vivo imaging of butterfly wing development: revealing the cellular dynamics of the pupal wing tissue.

Science.gov (United States)

Iwata, Masaki; Ohno, Yoshikazu; Otaki, Joji M

2014-01-01

Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the cellular dynamics of living

Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data

NARCIS (Netherlands)

Ramakers, Christian; Ruijter, Jan M.; Deprez, Ronald H. Lekanne; Moorman, Antoon F. M.

2003-01-01

Quantification of mRNAs using real-time polymerase chain reaction (PCR) by monitoring the product formation with the fluorescent dye SYBR Green I is being extensively used in neurosciences, developmental biology, and medical diagnostics. Most PCR data analysis procedures assume that the PCR

Selecting a set of housekeeping genes for quantitative real-time PCR in normal and tetraploid haemocytes of soft-shell clams, Mya arenaria.

Science.gov (United States)

Siah, A; Dohoo, C; McKenna, P; Delaporte, M; Berthe, F C J

2008-09-01

The transcripts involved in the molecular mechanisms of haemic neoplasia in relation to the haemocyte ploidy status of the soft-shell clam, Mya arenaria, have yet to be identified. For this purpose, real-time quantitative RT-PCR constitutes a sensitive and efficient technique, which can help determine the gene expression involved in haemocyte tetraploid status in clams affected by haemic neoplasia. One of the critical steps in comparing transcription profiles is the stability of selected housekeeping genes, as well as an accurate normalization. In this study, we selected five reference genes, S18, L37, EF1, EF2 and actin, generally used as single control genes. Their expression was analyzed by real-time quantitative RT-PCR at different levels of haemocyte ploidy status in order to select the most stable genes. Using the geNorm software, our results showed that L37, EF1 and S18 represent the most stable gene expressions related to various ploidy status ranging from 0 to 78% of tetraploid haemocytes in clams sampled in North River (Prince Edward Island, Canada). However, actin gene expression appeared to be highly regulated. Hence, using it as a housekeeping gene in tetraploid haemocytes can result in inaccurate data. To compare gene expression levels related to haemocyte ploidy status in Mya arenaria, using L37, EF1 and S18 as housekeeping genes for accurate normalization is therefore recommended.

Real-time Social Internet Data to Guide Forecasting Models

Energy Technology Data Exchange (ETDEWEB)

Del Valle, Sara Y. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2016-09-20

Our goal is to improve decision support by monitoring and forecasting events using social media, mathematical models, and quantifying model uncertainty. Our approach is real-time, data-driven forecasts with quantified uncertainty: Not just for weather anymore. Information flow from human observations of events through an Internet system and classification algorithms is used to produce quantitatively uncertain forecast. In summary, we want to develop new tools to extract useful information from Internet data streams, develop new approaches to assimilate real-time information into predictive models, validate approaches by forecasting events, and our ultimate goal is to develop an event forecasting system using mathematical approaches and heterogeneous data streams.

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR.

Science.gov (United States)

Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

2011-01-01

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch.

Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting the groEL Gene

Science.gov (United States)

Junick, Jana

2012-01-01

Quantitative real-time PCR assays targeting the groEL gene for the specific enumeration of 12 human fecal Bifidobacterium species were developed. The housekeeping gene groEL (HSP60 in eukaryotes) was used as a discriminative marker for the differentiation of Bifidobacterium adolescentis, B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. dentium, B. gallicum, B. longum, B. pseudocatenulatum, B. pseudolongum, and B. thermophilum. The bifidobacterial chromosome contains a single copy of the groEL gene, allowing the determination of the cell number by quantification of the groEL copy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a given Bifidobacterium species. Independent of the Bifidobacterium species tested, the proportion of groEL copies recovered from fecal samples spiked with 5 to 9 log10 cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10 groEL copies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3 Bifidobacterium species and B. longum frequently detected. The predominant species in infant and adult fecal samples were B. breve and B. adolescentis, respectively. It was possible to distinguish B. catenulatum and B. pseudocatenulatum. We conclude that the groEL gene is a suitable molecular marker for the specific and accurate quantification of human fecal Bifidobacterium species by real-time PCR. PMID:22307308

Applying real-time quantitative PCR to diagnosis of freemartin in Holstein cattle by quantifying SRY gene: a comparison experiment

Directory of Open Access Journals (Sweden)

Qinghua Qiu

2018-04-01

Full Text Available Background Freemartinism generally occurs in female offspring of dizygotic twins in a mixed-sex pregnancy. Most bovine heterosexual twin females are freemartins. However, about 10% of bovine heterosexual twin females are fertile. Farmers mostly cull bovine fertile heterosexual twin females due to the lack of a practical diagnostic approach. Culling of such animals results in economic and genetic-material losses both for dairy and beef industry. Methods In this study, a comparative test, including qualitative detection of SRY gene by polymerase chain reaction (PCR, quantitative detection of relative content of SRY by real-time quantitative polymerase chain reaction (qPCR, and quantitative detection of H-Y antigen, was performed to establish the most accurate diagnosis for freemartin. Twelve Holstein heterosexual twin females were used in this study, while three normal Holstein bulls and three normal Holstein cows were used as a positive and negative control, respectively. Results Polymerase chain reaction results revealed that SRY gene were absent in three heterosexual twin females and only two of them were verified as fertile in later age. The qPCR results showed that relative content of SRY was more than 14.2% in freemartins and below 0.41% in fertile heterosexual twin females. The H-Y antigen test showed no significant numerical difference between freemartin and fertile heterosexual twin female. Discussion Our results show that relative content of SRY quantified by qPCR is a better detection method for diagnosis of freemartin in Holstein cattle as compare to qualitative detection of SRY gene by PCR or quantitative detection of H-Y antigen. To the authors’ knowledge, this is the first time we applied qPCR to diagnosing freemartin by quantifying SRY gene and got relative SRY content of each freemartin and fertile heterosexual twin female. We concluded that low-level of SRY would not influence fertility of bovine heterosexual twin female.

OSL Based Anthropomorphic Phantom and Real-Time Organ Dosimetry

Energy Technology Data Exchange (ETDEWEB)

David E. Hintenlang, Ph.D

2009-02-10

The overall objective of this project was the development of a dosimetry system that provides the direct measurement of organ does in real-time with a sensitivity that makes it an effective tool for applications in a wide variety of health physics applications. The system included the development of a real-time readout system for fiber optic coupled (FOC) dosimeters that is integrated with a state-of-art anthropomorphic phantom to provide instantaneous measures of organ doses throughout the phantom. The small size of the FOC detectors and optical fibers allow the sensitive volume of the detector to be located at organ centroids (or multiple locations distributed through the organ) within a tissue equivalent, anthropomorphic phantom without perturbing the tissue equivalent features of the phantom. The developed phantom/dosimetry system can be used in any environment where personnel may be exposed to gamma or x-ray radiations to provide the most accurate determinations of organ and effective doses possible to date.

OSL Based Anthropomorphic Phantom and Real-Time Organ Dosimetry

International Nuclear Information System (INIS)

Hintenlang, David E.

2009-01-01

The overall objective of this project was the development of a dosimetry system that provides the direct measurement of organ doses in real-time with a sensitivity that makes it an effective tool for applications in a wide variety of health physics applications. The system included the development of a real-time readout system for fiber optic coupled (FOC) dosimeters that is integrated with a state-of-art anthropomorphic phantom to provide instantaneous measures of organ doses throughout the phantom. The small size of the FOC detectors and optical fibers allow the sensitive volume of the detector to be located at organ centroids (or multiple locations distributed through the organ) within a tissue equivalent, anthropomorphic phantom without perturbing the tissue equivalent features of the phantom. The developed phantom/dosimetry system can be used in any environment where personnel may be exposed to gamma or x-ray radiations to provide the most accurate determinations of organ and effective doses possible to date

Quantitatively differentiating microstructural variations of skeletal muscle tissues by multispectral Mueller matrix imaging

Science.gov (United States)

Dong, Yang; He, Honghui; He, Chao; Ma, Hui

2016-10-01

Polarized light is sensitive to the microstructures of biological tissues and can be used to detect physiological changes. Meanwhile, spectral features of the scattered light can also provide abundant microstructural information of tissues. In this paper, we take the backscattering polarization Mueller matrix images of bovine skeletal muscle tissues during the 24-hour experimental time, and analyze their multispectral behavior using quantitative Mueller matrix parameters. In the processes of rigor mortis and proteolysis of muscle samples, multispectral frequency distribution histograms (FDHs) of the Mueller matrix elements can reveal rich qualitative structural information. In addition, we analyze the temporal variations of the sample using the multispectral Mueller matrix transformation (MMT) parameters. The experimental results indicate that the different stages of rigor mortis and proteolysis for bovine skeletal muscle samples can be judged by these MMT parameters. The results presented in this work show that combining with the multispectral technique, the FDHs and MMT parameters can characterize the microstructural variation features of skeletal muscle tissues. The techniques have the potential to be used as tools for quantitative assessment of meat qualities in food industry.

Securing Real-Time Sessions in an IMS-Based Architecture

Science.gov (United States)

Cennamo, Paolo; Fresa, Antonio; Longo, Maurizio; Postiglione, Fabio; Robustelli, Anton Luca; Toro, Francesco

The emerging all-IP mobile network infrastructures based on 3rd Generation IP Multimedia Subsystem philosophy are characterised by radio access technology independence and ubiquitous connectivity for mobile users. Currently, great focus is being devoted to security issues since most of the security threats presently affecting the public Internet domain, and the upcoming ones as well, are going to be suffered by mobile users in the years to come. While a great deal of research activity, together with standardisation efforts and experimentations, is carried out on mechanisms for signalling protection, very few integrated frameworks for real-time multimedia data protection have been proposed in a context of IP Multimedia Subsystem, and even fewer experimental results based on testbeds are available. In this paper, after a general overview of the security issues arising in an advanced IP Multimedia Subsystem scenario, a comprehensive infrastructure for real-time multimedia data protection, based on the adoption of the Secure Real-Time Protocol, is proposed; then, the development of a testbed incorporating such functionalities, including mechanisms for key management and cryptographic context transfer, and allowing the setup of Secure Real-Time Protocol sessions is presented; finally, experimental results are provided together with quantitative assessments and comparisons of system performances for audio sessions with and without the adoption of the Secure Real-Time Protocol framework.

Quantitative frequency-domain fluorescence spectroscopy in tissues and tissue-like media

Science.gov (United States)

Cerussi, Albert Edward

1999-09-01

In the never-ending quest for improved medical technology at lower cost, modern near-infrared optical spectroscopy offers the possibility of inexpensive technology for quantitative and non-invasive diagnoses. Hemoglobin is the dominant chromophore in the 700-900 nm spectral region and as such it allows for the optical assessment of hemoglobin concentration and tissue oxygenation by absorption spectroscopy. However, there are many other important physiologically relevant compounds or physiological states that cannot be effectively sensed via optical methods because of poor optical contrast. In such cases, contrast enhancements are required. Fluorescence spectroscopy is an attractive component of optical tissue spectroscopy. Exogenous fluorophores, as well as some endogenous ones, may furnish the desperately needed sensitivity and specificity that is lacking in near-infrared optical tissue spectroscopy. The main focus of this thesis was to investigate the generation and propagation of fluorescence photons inside tissues and tissue-like media (i.e., scattering dominated media). The standard concepts of fluorescence spectroscopy have been incorporated into a diffusion-based picture that is sometimes referred to as photon migration. The novelty of this work lies in the successful quantitative recovery of fluorescence lifetimes, absolute fluorescence quantum yields, fluorophore concentrations, emission spectra, and both scattering and absorption coefficients at the emission wavelength from a tissue-like medium. All of these parameters are sensitive to the fluorophore local environment and hence are indicators of the tissue's physiological state. One application demonstrating the capabilities of frequency-domain lifetime spectroscopy in tissue-like media is a study of the binding of ethidium bromide to bovine leukocytes in fresh milk. Ethidium bromide is a fluorescent dye that is commonly used to label DNA, and hence visualize chromosomes in cells. The lifetime of

Review of Real-Time 3-Dimensional Image Guided Radiation Therapy on Standard-Equipped Cancer Radiation Therapy Systems: Are We at the Tipping Point for the Era of Real-Time Radiation Therapy?

Science.gov (United States)

Keall, Paul J; Nguyen, Doan Trang; O'Brien, Ricky; Zhang, Pengpeng; Happersett, Laura; Bertholet, Jenny; Poulsen, Per R

2018-04-14

To review real-time 3-dimensional (3D) image guided radiation therapy (IGRT) on standard-equipped cancer radiation therapy systems, focusing on clinically implemented solutions. Three groups in 3 continents have clinically implemented novel real-time 3D IGRT solutions on standard-equipped linear accelerators. These technologies encompass kilovoltage, combined megavoltage-kilovoltage, and combined kilovoltage-optical imaging. The cancer sites treated span pelvic and abdominal tumors for which respiratory motion is present. For each method the 3D-measured motion during treatment is reported. After treatment, dose reconstruction was used to assess the treatment quality in the presence of motion with and without real-time 3D IGRT. The geometric accuracy was quantified through phantom experiments. A literature search was conducted to identify additional real-time 3D IGRT methods that could be clinically implemented in the near future. The real-time 3D IGRT methods were successfully clinically implemented and have been used to treat more than 200 patients. Systematic target position shifts were observed using all 3 methods. Dose reconstruction demonstrated that the delivered dose is closer to the planned dose with real-time 3D IGRT than without real-time 3D IGRT. In addition, compromised target dose coverage and variable normal tissue doses were found without real-time 3D IGRT. The geometric accuracy results with real-time 3D IGRT had a mean error of real-time 3D IGRT methods using standard-equipped radiation therapy systems that could also be clinically implemented. Multiple clinical implementations of real-time 3D IGRT on standard-equipped cancer radiation therapy systems have been demonstrated. Many more approaches that could be implemented were identified. These solutions provide a pathway for the broader adoption of methods to make radiation therapy more accurate, impacting tumor and normal tissue dose, margins, and ultimately patient outcomes. Copyright © 2018

Detection of tumor markers in prostate cancer and comparison of sensitivity between real time and nested PCR.

Science.gov (United States)

Matsuoka, Takayuki; Shigemura, Katsumi; Yamamichi, Fukashi; Fujisawa, Masato; Kawabata, Masato; Shirakawa, Toshiro

2012-06-27

The objective of this study is to investigate and compare the sensitivity in conventional PCR, quantitative real time PCR, nested PCR and western blots for detection of prostate cancer tumor markers using prostate cancer (PCa) cells. We performed conventional PCR, quantitative real time PCR, nested PCR, and western blots using 5 kinds of PCa cells. Prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and androgen receptor (AR) were compared for their detection sensitivity by real time PCR and nested PCR. In real time PCR, there was a significant correlation between cell number and the RNA concentration obtained (R(2)=0.9944) for PSA, PSMA, and AR. We found it possible to detect these markers from a single LNCaP cell in both real time and nested PCR. By comparison, nested PCR reached a linear curve in fewer PCR cycles than real time PCR, suggesting that nested PCR may offer PCR results more quickly than real time PCR. In conclusion, nested PCR may offer tumor maker detection in PCa cells more quickly (with fewer PCR cycles) with the same high sensitivity as real time PCR. Further study is necessary to establish and evaluate the best tool for PCa tumor marker detection.

[Comparison of two quantitative methods of endobronchial ultrasound real-time elastography for evaluating intrathoracic lymph nodes].

Science.gov (United States)

Mao, X W; Yang, J Y; Zheng, X X; Wang, L; Zhu, L; Li, Y; Xiong, H K; Sun, J Y

2017-06-12

Objective: To compare the clinical value of two quantitative methods in analyzing endobronchial ultrasound real-time elastography (EBUS-RTE) images for evaluating intrathoracic lymph nodes. Methods: From January 2014 to April 2014, EBUS-RTE examination was performed in patients who received EBUS-TBNA examination in Shanghai Chest Hospital. Each intrathoracic lymph node had a selected EBUS-RTE image. Stiff area ratio and mean hue value of region of interest (ROI) in each image were calculated respectively. The final diagnosis of lymph node was based on the pathologic/microbiologic results of EBUS-TBNA, pathologic/microbiologic results of other examinations and clinical following-up. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy were evaluated for distinguishing malignant and benign lesions. Results: Fifty-six patients and 68 lymph nodes were enrolled in this study, of which 35 lymph nodes were malignant and 33 lymph nodes were benign. The stiff area ratio and mean hue value of benign and malignant lesions were 0.32±0.29, 0.62±0.20 and 109.99±28.13, 141.62±17.52, respectively, and statistical differences were found in both of those two methods ( t =-5.14, P methods can be used for analyzing EBUS-RTE images quantitatively, having the value of differentiating benign and malignant intrathoracic lymph nodes, and the stiff area ratio is better than the mean hue value between the two methods.

Friction coefficient of skin in real-time.

Science.gov (United States)

Sivamani, Raja K; Goodman, Jack; Gitis, Norm V; Maibach, Howard I

2003-08-01

Friction studies are useful in quantitatively investigating the skin surface. Previous studies utilized different apparatuses and materials for these investigations but there was no real-time test parameter control or monitoring. Our studies incorporated the commercially available UMT Series Micro-Tribometer, a tribology instrument that permits real-time monitoring and calculation of the important parameters in friction studies, increasing the accuracy over previous tribology and friction measurement devices used on skin. Our friction tests were performed on four healthy volunteers and on abdominal skin samples. A stainless steel ball was pressed on to the skin with at a pre-set load and then moved across the skin at a constant velocity of 5 mm/min. The UMT continuously monitored the friction force of the skin and the normal force of the ball to calculate the friction coefficient in real-time. Tests investigated the applicability of Amonton's law, the impact of increased and decreased hydration, and the effect of the application of moisturizers. The friction coefficient depends on the normal load applied, and Amonton's law does not provide an accurate description for the skin surface. Application of water to the skin increased the friction coefficient and application of isopropyl alcohol decreased it. Fast acting moisturizers immediately increased the friction coefficient, but did not have the prolonged effect of the slow, long lasting moisturizers. The UMT is capable of making real-time measurements on the skin and can be used as an effective tool to study friction properties. Results from the UMT measurements agree closely with theory regarding the skin surface.

Real-Time Vision-Based Stiffness Mapping †.

Science.gov (United States)

Faragasso, Angela; Bimbo, João; Stilli, Agostino; Wurdemann, Helge Arne; Althoefer, Kaspar; Asama, Hajime

2018-04-26

This paper presents new findings concerning a hand-held stiffness probe for the medical diagnosis of abnormalities during palpation of soft-tissue. Palpation is recognized by the medical community as an essential and low-cost method to detect and diagnose disease in soft-tissue. However, differences are often subtle and clinicians need to train for many years before they can conduct a reliable diagnosis. The probe presented here fills this gap providing a means to easily obtain stiffness values of soft tissue during a palpation procedure. Our stiffness sensor is equipped with a multi degree of freedom (DoF) Aurora magnetic tracker, allowing us to track and record the 3D position of the probe whilst examining a tissue area, and generate a 3D stiffness map in real-time. The stiffness probe was integrated in a robotic arm and tested in an artificial environment representing a good model of soft tissue organs; the results show that the sensor can accurately measure and map the stiffness of a silicon phantom embedded with areas of varying stiffness.

Real-Time Vision-Based Stiffness Mapping †

Directory of Open Access Journals (Sweden)

Angela Faragasso

2018-04-01

Full Text Available This paper presents new findings concerning a hand-held stiffness probe for the medical diagnosis of abnormalities during palpation of soft-tissue. Palpation is recognized by the medical community as an essential and low-cost method to detect and diagnose disease in soft-tissue. However, differences are often subtle and clinicians need to train for many years before they can conduct a reliable diagnosis. The probe presented here fills this gap providing a means to easily obtain stiffness values of soft tissue during a palpation procedure. Our stiffness sensor is equipped with a multi degree of freedom (DoF Aurora magnetic tracker, allowing us to track and record the 3D position of the probe whilst examining a tissue area, and generate a 3D stiffness map in real-time. The stiffness probe was integrated in a robotic arm and tested in an artificial environment representing a good model of soft tissue organs; the results show that the sensor can accurately measure and map the stiffness of a silicon phantom embedded with areas of varying stiffness.

Quantification of algal endosymbionts (Symbiodinium) in coral tissue using real-time PCR

NARCIS (Netherlands)

Mieog, J. C.; Van Oppen, M. J. H.; Berkelmans, R.; Stam, W. T.; Olsen, J. L.

Understanding the flexibility of the endosymbioses between scleractinian corals and single-cell algae of the genus Symbiodinium will provide valuable insights into the future of coral reefs. Here, a real-time polymerase chain reaction (PCR) assay is presented to accurately determine the cell

Application of reverse transcription-PCR and real-time PCR in nanotoxicity research.

Science.gov (United States)

Mo, Yiqun; Wan, Rong; Zhang, Qunwei

2012-01-01

Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq(®) DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix.

Development of a quantitative PCR assay for monitoring Streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia.

Science.gov (United States)

Su, Y-L; Feng, J; Li, Y-W; Bai, J-S; Li, A-X

2016-02-01

Streptococcus agalactiae has become one of the most important emerging pathogens in the aquaculture industry and has resulted in large economic losses for tilapia farms in China. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real-time polymerase chain reactions (qPCRs) after optimization of the annealing temperature. The primer pair IGS-s/IGS-a, which targets the 16S-23S rRNA intergenic spacer region, was finally chosen, having a detection limit of 8.6 copies of S. agalactiae DNA in a 20 μL reaction mixture. Bacterial tissue tropism was demonstrated by qPCR in Oreochromis niloticus 5 days post-injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, followed by moderately high levels in kidney, heart, spleen, intestines, and eye. Significantly lower bacterial loads were observed in muscle, gill and liver. In addition, significantly lower bacterial loads were observed in the brain of convalescent O. niloticus 14 days post-injection with several different S. agalactiae strains. The qPCR for the detection of S. agalactiae developed in this study provides a quantitative tool for investigating bacterial tissue tropism in infected fish, as well as for monitoring bacterial colonization in convalescent fish. © 2015 John Wiley & Sons Ltd.

Detection of Tumor Markers in Prostate Cancer and Comparison of Sensitivity between Real Time and Nested PCR

OpenAIRE

Matsuoka, Takayuki; Shigemura, Katsumi; Yamamichi, Fukashi; Fujisawa, Masato; Kawabata, Masato; Shirakawa, Toshiro

2012-01-01

The objective of this study is to investigate and compare the sensitivity in conventional PCR, quantitative real time PCR, nested PCR and western blots for detection of prostate cancer tumor markers using prostate cancer (PCa) cells. We performed conventional PCR, quantitative real time PCR, nested PCR, and western blots using 5 kinds of PCa cells. Prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and androgen receptor (AR) were compared for their detection sensitivi...

Response of a diuron-degrading community to diuron exposure assessed by real-time quantitative PCR monitoring of phenylurea hydrolase A and B encoding genes

OpenAIRE

Pesce , S.; Beguet , J.; Rouard , N.; Devers Lamrani , M.; Martin Laurent , F.

2013-01-01

A real-time quantitative PCR method was developed to detect and quantify phenlylurea hydrolase genes' (puhA and puhB) sequences from environmental DNA samples to assess diuron-degrading genetic potential in some soil and sediment microbial communities. In the soil communities, mineralization rates (determined with [ring-14C]-labeled diuron) were linked to diuron-degrading genetic potentials estimated from puhB number copies, which increased following repeated diuron treatments. In the sedimen...

Real-time shadows

CERN Document Server

Eisemann, Elmar; Assarsson, Ulf; Wimmer, Michael

2011-01-01

Important elements of games, movies, and other computer-generated content, shadows are crucial for enhancing realism and providing important visual cues. In recent years, there have been notable improvements in visual quality and speed, making high-quality realistic real-time shadows a reachable goal. Real-Time Shadows is a comprehensive guide to the theory and practice of real-time shadow techniques. It covers a large variety of different effects, including hard, soft, volumetric, and semi-transparent shadows.The book explains the basics as well as many advanced aspects related to the domain

Real-time detection of TDP1 activity using a fluorophore-quencher coupled DNA-biosensor

DEFF Research Database (Denmark)

Jensen, Pia Wrensted; Falconi, Mattia; Kristoffersen, Emil Laust

2013-01-01

structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only “signal amplification” the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1......Real-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very...... simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples...

Dependable Real-Time Systems

Science.gov (United States)

1991-09-30

0196 or 413 545-0720 PI E-mail Address: krithi@nirvan.cs.umass.edu, stankovic(ocs.umass.edu Grant or Contract Title: Dependable Real - Time Systems Grant...Dependable Real - Time Systems " Grant or Contract Number: N00014-85-k-0398 L " Reporting Period: 1 Oct 87 - 30 Sep 91 , 2. Summary of Accomplishments ’ 2.1 Our...in developing a sound approach to scheduling tasks in complex real - time systems , (2) developed a real-time operating system kernel, a preliminary

Real time speckle monitoring to control retinal photocoagulation

Science.gov (United States)

Bliedtner, Katharina; Seifert, Eric; Brinkmann, Ralf

2017-07-01

Photocoagulation is a treatment modality for several retinal diseases. Intra- and inter-individual variations of the retinal absorption as well as ocular transmission and light scattering makes it impossible to achieve a uniform effective exposure with one set of laser parameters. To guarantee a uniform damage throughout the therapy a real-time control is highly requested. Here, an approach to realize a real-time optical feedback using dynamic speckle analysis in-vivo is presented. A 532 nm continuous wave Nd:YAG laser is used for coagulation. During coagulation, speckle dynamics are monitored by a coherent object illumination using a 633 nm diode laser and analyzed by a CMOS camera with a frame rate up to 1 kHz. An algorithm is presented that can discriminate between different categories of retinal pigment epithelial damage ex-vivo in enucleated porcine eyes and that seems to be robust to noise in-vivo. Tissue changes in rabbits during retinal coagulation could be observed for different lesion strengths. This algorithm can run on a FPGA and is able to calculate a feedback value which is correlated to the thermal and coagulation induced tissue motion and thus the achieved damage.

LabVIEW Real-Time

CERN Multimedia

CERN. Geneva; Flockhart, Ronald Bruce; Seppey, P

2003-01-01

With LabVIEW Real-Time, you can choose from a variety of RT Series hardware. Add a real-time data acquisition component into a larger measurement and automation system or create a single stand-alone real-time solution with data acquisition, signal conditioning, motion control, RS-232, GPIB instrumentation, and Ethernet connectivity. With the various hardware options, you can create a system to meet your precise needs today, while the modularity of the system means you can add to the solution as your system requirements grow. If you are interested in Reliable and Deterministic systems for Measurement and Automation, you will profit from this seminar. Agenda: Real-Time Overview LabVIEW RT Hardware Platforms - Linux on PXI Programming with LabVIEW RT Real-Time Operating Systems concepts Timing Applications Data Transfer

Development of Quantitative Competitive PCR and Absolute Based Real-Time PCR Assays for Quantification of The Butyrate Producing Bacterium: Butyrivibrio fibrisolvens

Directory of Open Access Journals (Sweden)

Mojtaba Tahmoorespur

2016-04-01

Full Text Available Introduction Butyrivibrio fibrisolvens strains are presently recognized as the major butyrate-producing bacteria found in the rumen and digestive track of many animals and also in the human gut. In this study we reported the development of two DNA based techniques, quantitative competitive (QC PCR and absolute based Real-Time PCR, for enumerating Butyrivibrio fibrisolvens strains. Despite the recent introduction of real-time PCR method for the rapid quantification of the target DNA sequences, use of quantitative competitive PCR (QC-PCR technique continues to play an important role in nucleic acid quantification since it is more cost effective. The procedure relies on the co-amplification of the sequence of interest with a serially diluted synthetic DNA fragment of the known concentration (competitor, using the single set primers. A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR. It monitors the amplification of a targeted DNA molecule during the PCR. Materials and Methods At first reported species-specific primers targeting the 16S rDNA region of the bacterium Butyrivibrio fibrisolvens were used for amplifying a 213 bp fragment. A DNA competitor differing by 50 bp in length from the 213 bp fragment was constructed and cloned into pTZ57R/T vector. The competitor was quantified by NanoDrop spectrophotometer and serially diluted and co-amplified by PCR with total extracted DNA from rumen fluid samples. PCR products were quantified by photographing agarose gels and analyzed with Image J software and the amount of amplified target DNA was log plotted against the amount of amplified competitor. Coefficient of determination (R2 was used as a criterion of methodology precision. For developing the Real-time PCR technique, the 213 bp fragment was amplified and cloned into pTZ57R/T was used to draw a standard curve. Results and Discussion The specific primers of Butyrivibrio

Concepts of real time and semi-real time material control

International Nuclear Information System (INIS)

Lovett, J.E.

1975-01-01

After a brief consideration of the traditional material balance accounting on an MBA basis, this paper explores the basic concepts of real time and semi-real time material control, together with some of the major problems to be solved. Three types of short-term material control are discussed: storage, batch processing, and continuous processing. (DLC)

Effectiveness of Quantitative Real Time PCR in Long-Term Follow-up of Chronic Myeloid Leukemia Patients.

Science.gov (United States)

Savasoglu, Kaan; Payzin, Kadriye Bahriye; Ozdemirkiran, Fusun; Berber, Belgin

2015-08-01

To determine the use of the Quantitative Real Time PCR (RQ-PCR) assay follow-up with Chronic Myeloid Leukemia (CML) patients. Cross-sectional observational. Izmir Ataturk Education and Research Hospital, Izmir, Turkey, from 2009 to 2013. Cytogenetic, FISH, RQ-PCR test results from 177 CMLpatients' materials selected between 2009 - 2013 years was set up for comparison analysis. Statistical analysis was performed to compare between FISH, karyotype and RQ-PCR results of the patients. Karyotyping and FISH specificity and sensitivity rates determined by ROC analysis compared with RQ-PCR results. Chi-square test was used to compare test failure rates. Sensitivity and specificity values were determined for karyotyping 17.6 - 98% (p=0.118, p > 0.05) and for FISH 22.5 - 96% (p=0.064, p > 0.05) respectively. FISH sensitivity was slightly higher than karyotyping but there was calculated a strong correlation between them (p 0.05); however, karyotyping and FISH test failure rate was statistically significant (p < 0.001). Besides, the situation needed for karyotype analysis, RQ-PCR assay can be used alone in the follow-up of CMLdisease.

Real Time Systems

DEFF Research Database (Denmark)

Christensen, Knud Smed

2000-01-01

Describes fundamentals of parallel programming and a kernel for that. Describes methods for modelling and checking parallel problems. Real time problems.......Describes fundamentals of parallel programming and a kernel for that. Describes methods for modelling and checking parallel problems. Real time problems....

Real time expert systems

International Nuclear Information System (INIS)

Asami, Tohru; Hashimoto, Kazuo; Yamamoto, Seiichi

1992-01-01

Recently, aiming at the application to the plant control for nuclear reactors and traffic and communication control, the research and the practical use of the expert system suitable to real time processing have become conspicuous. In this report, the condition for the required function to control the object that dynamically changes within a limited time is presented, and the technical difference between the real time expert system developed so as to satisfy it and the expert system of conventional type is explained with the actual examples and from theoretical aspect. The expert system of conventional type has the technical base in the problem-solving equipment originating in STRIPS. The real time expert system is applied to the fields accompanied by surveillance and control, to which conventional expert system is hard to be applied. The requirement for the real time expert system, the example of the real time expert system, and as the techniques of realizing real time processing, the realization of interruption processing, dispersion processing, and the mechanism of maintaining the consistency of knowledge are explained. (K.I.)

Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR.

Science.gov (United States)

Endo, Noriyuki; Sato, Kana; Matsumura, Kiyotaka; Yoshimura, Erina; Odaka, Yukiko; Nogata, Yasuyuki

2010-11-01

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.

Real-time porphyrin detection in plaque and caries: a case study

Science.gov (United States)

Timoshchuk, Mari-Alina I.; Ridge, Jeremy S.; Rugg, Amanda L.; Nelson, Leonard Y.; Kim, Amy S.; Seibel, Eric J.

2015-02-01

An ultrathin scanning fiber endoscope, originally developed for cancer diagnosis, was used in a case study to locate plaque and caries. The imaging system incorporated software mitigation of background auto-fluorescence (AF). In conventional fluorescence imaging, varying AF across a tooth surface can mask low-level porphyrin signals. Laser-induced auto-fluorescence signals of dental tissue excited using a 405-nm laser typically produce fluorescence over a wavelength range extending from 440-nm to 750-nm. Anaerobic bacterial metabolism produces various porphyrin species (eg. protoporphyrin IX) that are located in carious enamel, dentin, gingivitis sites, and plaque. In our case study, these porphyrin deposits remained as long as one day after prophylaxis. Imaging the tooth surface using 405-nm excitation and subtracting the natural AF enhances the image contrast of low-level porphyrin deposits, which would otherwise be masked by the high background AF. In a case study, healthy tissues as well as sites of early and advanced caries formations were scanned for visual and quantitative signs of red fluorescence associated with porphyrin species using a background mitigation algorithm. Initial findings show increasing amplitudes of red fluorescence as caries severity increases from early to late stages. Sites of plaque accumulation also displayed red fluorescence similar to that found in carious dental tissue. The use of real-time background mitigation of natural dental AF can enhance the detection of low porphyrin concentrations that are indicators of early stage caries formation.

Development and application of an oligonucleotide microarray and real-time quantitative PCR for detection of wastewater bacterial pathogens

Energy Technology Data Exchange (ETDEWEB)

Lee, Dae-Young [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada)], E-mail: daeyoung.lee@ec.gc.ca; Lauder, Heather; Cruwys, Heather; Falletta, Patricia [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada); Beaudette, Lee A. [Environmental Science and Technology Centre, Environment Canada, 335 River Road South, Ottawa, Ontario, K1A 0H3 (Canada)], E-mail: lee.beaudette@ec.gc.ca

2008-07-15

Conventional microbial water quality test methods are well known for their technical limitations, such as lack of direct pathogen detection capacity and low throughput capability. The microarray assay has recently emerged as a promising alternative for environmental pathogen monitoring. In this study, bacterial pathogens were detected in municipal wastewater using a microarray equipped with short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes have been designed against 38 species, 4 genera, and 1 family of pathogens. The detection sensitivity of the microarray for a waterborne pathogen Aeromonas hydrophila was determined to be approximately 1.0% of the total DNA, or approximately 10{sup 3}A. hydrophila cells per sample. The efficacy of the DNA microarray was verified in a parallel study where pathogen genes and E. coli cells were enumerated using real-time quantitative PCR (qPCR) and standard membrane filter techniques, respectively. The microarray and qPCR successfully detected multiple wastewater pathogen species at different stages of the disinfection process (i.e. secondary effluents vs. disinfected final effluents) and at two treatment plants employing different disinfection methods (i.e. chlorination vs. UV irradiation). This result demonstrates the effectiveness of the DNA microarray as a semi-quantitative, high throughput pathogen monitoring tool for municipal wastewater.

Recombinant plasmid-based quantitative Real-Time PCR analysis of Salmonella enterica serotypes and its application to milk samples.

Science.gov (United States)

Gokduman, Kurtulus; Avsaroglu, M Dilek; Cakiris, Aris; Ustek, Duran; Gurakan, G Candan

2016-03-01

The aim of the current study was to develop, a new, rapid, sensitive and quantitative Salmonella detection method using a Real-Time PCR technique based on an inexpensive, easy to produce, convenient and standardized recombinant plasmid positive control. To achieve this, two recombinant plasmids were constructed as reference molecules by cloning the two most commonly used Salmonella-specific target gene regions, invA and ttrRSBC. The more rapid detection enabled by the developed method (21 h) compared to the traditional culture method (90 h) allows the quantitative evaluation of Salmonella (quantification limits of 10(1)CFU/ml and 10(0)CFU/ml for the invA target and the ttrRSBC target, respectively), as illustrated using milk samples. Three advantages illustrated by the current study demonstrate the potential of the newly developed method to be used in routine analyses in the medical, veterinary, food and water/environmental sectors: I--The method provides fast analyses including the simultaneous detection and determination of correct pathogen counts; II--The method is applicable to challenging samples, such as milk; III--The method's positive controls (recombinant plasmids) are reproducible in large quantities without the need to construct new calibration curves. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I

DEFF Research Database (Denmark)

Marcussen, Lærke Bay; Jepsen, Morten Leth; Kristoffersen, Emil Laust

2013-01-01

Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I....... The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor....... The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented...

Evaluation of real-time operating system for small-scale embedded systems

International Nuclear Information System (INIS)

Dayang Norhayati Abang Jawawi; Rosbi Mamat

1999-01-01

In this paper, the performance of some real-time operating systems for small-scale embedded systems are evaluated based on some criteria. The evaluation is performed qualitatively and quantitatively. The evaluation results based on a case study on an engineering application will be presented. (author)

Decreased expression of transient receptor potential channels in cerebral vascular tissue from patients after hypertensive intracerebral hemorrhage

DEFF Research Database (Denmark)

Thilo, Florian; Suess, Olaf; Liu, Ying

2011-01-01

, TRPC5, TRPC6, TRPM4, TRPM6, and TRPM7 channels were detected in cerebral vascular tissue by quantitative real-time RT-PCR. Control cerebral vascular tissue was obtained from normotensive patients who underwent neurosurgical operation because of brain tumor. To examine a possible relation between...

Detection and identification of Rift Valley fever virus in mosquito vectors by quantitative real-time PCR.

Science.gov (United States)

Mwaengo, D; Lorenzo, G; Iglesias, J; Warigia, M; Sang, R; Bishop, R P; Brun, A

2012-10-01

Diagnostic methods allowing for rapid identification of pathogens are crucial for controlling and preventing dissemination after disease outbreaks as well as for use in surveillance programs. For arboviruses, detection of the presence of virus in their arthropod hosts is important for monitoring of viral activity and quantitative information is useful for modeling of transmission dynamics. In this study, molecular detection of Rift Valley fever virus (RVFV) in mosquito samples from the 2006 to 2007 East African outbreaks was performed using quantitative real-time PCR assay (qRT-PCR). Specific RVFV sequence-based primer/fluorogenic (TaqMan) probe sets were derived from the L and S RNA segments of the virus. Both primer-probe L and S segment-based combinations detected genomic RVFV sequences, with generally comparable levels of sensitivity. Viral loads from three mosquito species, Aedes mcintoshi, Aedes ochraceus and Mansonia uniformis were estimated and significant differences of between 5- and 1000-fold were detected between Ae. mcintoshi and M. uniformis using both the L and S primer-probe-based assays. The genetic relationships of the viral sequences in mosquito samples were established by partial M segment sequencing and assigned to the two previously described viral lineages defined by analysis of livestock isolates obtained during the 2006-2007 outbreak, confirming that similar viruses were present in both the vector and mammalian host. The data confirms the utility of qRT-PCR for identification and initial quantification of virus in mosquito samples during RVFV outbreaks. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

Directory of Open Access Journals (Sweden)

Hamel Christian

2003-01-01

Full Text Available Abstract Background X-linked ocular albinism type 1 (OA1 is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment. Results We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7–8, and a point mutation (310delG in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in OA1 gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA. Conclusion The identification of OA1 mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of OA1 exons with DMD exon as external standard performed on the LightCycler™ allows quick and accurate carrier-status assessment for at-risk females.

Real-Time Dynamic MLC Tracking for Intensity Modulated Arc Therapy

DEFF Research Database (Denmark)

Falk, Marianne

Motion management of intra-fraction tumour motion during radiotherapy treatment can be a challenging task in order to achieve tumour control as well as minimizing the dose to the surrounding healthy tissue. Real-time dynamic multileaf collimator (MLC) tracking is a novel method for intra-fraction...

Research on Control Method Based on Real-Time Operational Reliability Evaluation for Space Manipulator

Directory of Open Access Journals (Sweden)

Yifan Wang

2014-05-01

Full Text Available A control method based on real-time operational reliability evaluation for space manipulator is presented for improving the success rate of a manipulator during the execution of a task. In this paper, a method for quantitative analysis of operational reliability is given when manipulator is executing a specified task; then a control model which could control the quantitative operational reliability is built. First, the control process is described by using a state space equation. Second, process parameters are estimated in real time using Bayesian method. Third, the expression of the system's real-time operational reliability is deduced based on the state space equation and process parameters which are estimated using Bayesian method. Finally, a control variable regulation strategy which considers the cost of control is given based on the Theory of Statistical Process Control. It is shown via simulations that this method effectively improves the operational reliability of space manipulator control system.

[Detecting HB-1 Expression Level in Bone Marrow of Acute Leukemia Patients by Real-Time Fluorescence Quantitative RT-PCR].

Science.gov (United States)

Wang, Qing-Yun; Li, Yuan; Ji, Li; Liang, Ze-Yin; Liu, Wei; Ren, Han-Yun; Qiu, Zhi-Xiang

2018-02-01

To investigate the expression level of HB-1 gene in patients with acute lymphoblastic leukemia (ALL) and the significance of HB-1 gene in monitoring of minimal residual disease (MRD). The method of real-time fluorescence quantitative RT-PCR (Taqman probe) was established to detect the expression levels of HB-1 gene; then the sensitivity, specificity and repeatability of this assay were evaluated and verified. The HB-1 gene expression levels in bone marrow of 183 cases of ALL, 70 cases of acute myeloid leukemias (AML), 52 cases of non-malignant hematologic diseases and 24 healthy hematopoietic stem cell donors were detected. The correlation of HB-1 level with diagnosis and relapse was analyzed by detecting bone marrow samples of 33 B-ALL. The sensitivity of this assay reached the 10 -4 level. The coefficient of variation for inter-batch and inter-tube of HB-1 were 6.79% and 4.80%, respectively. It was found that HB-1 gene specifically expressed in acute B lymphoblastic leukemia. The median expression levels of HB-1 gene in newly diagnosed and relapsed B-ALL patients were statistically significantly higher than those in ALL in complete remission(CR), newly diagnosed T-ALL, newly diagnosed AML, non-malignant hematologic diseases, and healthy hematopoietic stem cell donors(33.0% vs 0.68%, 0.07%, 0.02%, 0.58% and 0, respectively) (P0.05). The expression level of HB-1 gene declined sharply when B-ALL patients reached complete remission (0-7.99%, with median level 0.68%), but increased when relapsed (7.69%, 8.08% and 484.0% in 3 relapsed samples), which was in accordance with results of flow cytometry. HB-1 gene specifically expressed in acute B lymphoblastic leukemia cells. The established real-time fluorescence quantitative RT-PCR assay shows good sensitivity, specificity and repeatability, thus, can be used as a biological marker in the clinical detection, monitoring MRD and predicting of early relapse for B-ALL patients.

A multimodal instrument for real-time in situ study of ultrasound and cavitation mediated drug delivery.

Science.gov (United States)

Bian, Shuning; Seth, Anjali; Daly, Dan; Carlisle, Robert; Stride, Eleanor

2017-03-01

The development of a multimodal instrument capable of real-time in situ measurements of cavitation activity and effect in tissue mimicking phantoms during ultrasound and cavitation mediated drug delivery experiments is described here. The instrument features an acoustic arm that can expose phantoms to high-intensity focused-ultrasound while measuring cavitation activity and an optical arm that monitors cavitation effect using confocal microscopy. This combination of modalities allows real-time in situ characterisation of drug delivery in tissue and tissue mimicking phantoms during ultrasound and cavitation mediated drug delivery experiments. A representative result, obtained with a tissue mimicking phantom and acoustically activated droplets, is presented here as a demonstration of the instrument's capabilities and potential applications.

Real-Time Mapping Spectroscopy on the Ground, in the Air, and in Space

Science.gov (United States)

Thompson, D. R.; Allwood, A.; Chien, S.; Green, R. O.; Wettergreen, D. S.

2016-12-01

Real-time data interpretation can benefit both remote in situ exploration and remote sensing. Basic analyses at the sensor can monitor instrument performance and reveal invisible science phenomena in real time. This promotes situational awareness for remote robotic explorers or campaign decision makers, enabling adaptive data collection, reduced downlink requirements, and coordinated multi-instrument observations. Fast analysis is ideal for mapping spectrometers providing unambiguous, quantitative geophysical measurements. This presentation surveys recent computational advances in real-time spectroscopic analysis for Earth science and planetary exploration. Spectral analysis at the sensor enables new operations concepts that significantly improve science yield. Applications include real-time detection of fugitive greenhouse emissions by airborne monitoring, real-time cloud screening and mineralogical mapping by orbital spectrometers, and adaptive measurement by the PIXL instrument on the Mars 2020 rover. Copyright 2016 California Institute of Technology. All Rights Reserved. We acknowledge support of the US Government, NASA, the Earth Science Division and Terrestrial Ecology program.

Development of a real-time RT-PCR assay for the simultaneous identification, quantitation and differentiation of avian metapneumovirus subtypes A and B.

Science.gov (United States)

Cecchinato, Mattia; Lupini, Caterina; Munoz Pogoreltseva, Olga Svetlana; Listorti, Valeria; Mondin, Alessandra; Drigo, Michele; Catelli, Elena

2013-01-01

In recent years, special attention has been paid to real-time polymerase chain reaction (PCR) for avian metapneumovirus (AMPV) diagnosis, due to its numerous advantages over classical PCR. A new multiplex quantitative real-time reverse transcription-PCR (qRT-PCR) with molecular beacon probe assay, designed to target the SH gene, was developed. The test was evaluated in terms of specificity, sensitivity and repeatability, and compared with conventional RT nested-PCR based on the G gene. All of the AMPV subtype A and B strains tested were amplified and specifically detected while no amplification occurred with other non-target bird respiratory pathogens. The detection limit of the assay was 10(-0.41) median infectious dose/ml and 10(1.15) median infectious dose/ml when the AMPV-B strain IT/Ty/B/Vr240/87 and the AMPV-A strain IT/Ty/A/259-01/03 were used, respectively, as templates. In all cases, the amplification efficiency was approximately 2 and the error values were 0.9375) between crossing point values and virus quantities, making the assay herein designed reliable for quantification. When the newly developed qRT-PCR was compared with a conventional RT nested-PCR, it showed greater sensitivity with RNA extracted from both positive controls and from experimentally infected birds. This assay can be effectively used for the detection, identification, differentiation and quantitation of AMPV subtype A or subtype B to assist in disease diagnosis and to carry out rapid surveillance with high levels of sensitivity and specificity.

Real-time simulation of soft tissue deformation and electrocautery procedures in laparoscopic rectal cancer radical surgery.

Science.gov (United States)

Sui, Yuan; Pan, Jun J; Qin, Hong; Liu, Hao; Lu, Yun

2017-12-01

Laparoscopic surgery (LS), also referred to as minimally invasive surgery, is a modern surgical technique which is widely applied. The fulcrum effect makes LS a non-intuitive motor skill with a steep learning curve. A hybrid model of tetrahedrons and a multi-layer triangular mesh are constructed to simulate the deformable behavior of the rectum and surrounding tissues in the Position-Based Dynamics (PBD) framework. A heat-conduction based electric-burn technique is employed to simulate the electrocautery procedure. The simulator has been applied for laparoscopic rectum cancer surgery training. From the experimental results, trainees can operate in real time with high degrees of stability and fidelity. A preliminary study was performed to evaluate the realism and usefulness. This prototype simulator has been tested and verified by colorectal surgeons through a pilot study. They believed both the visual and the haptic performance of the simulation are realistic and helpful to enhance laparoscopic skills. Copyright © 2017 John Wiley & Sons, Ltd.

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

Directory of Open Access Journals (Sweden)

Wilson Zoe A

2008-06-01

Full Text Available Abstract Background Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results We describe a real-time PCR technique employing attached universal duplex probes (AUDP, which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP and a complementary quenching probe (QP lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

Real-time TaqMan polymerase chain reaction to quantify the effects ...

African Journals Online (AJOL)

TaqMan polymerase chain reaction was developed to quantify the number of Bifidobacterium. We used this assay to detect genomic DNA of Bifidobacterium in the intestinal tract digesta of piglets, including duodenum, jejunum, ileum, cecum and colon. Our results indicated that, developed new real-time quantitative PCR ...

Visual detectability of elastic contrast in real-time ultrasound images

Science.gov (United States)

Miller, Naomi R.; Bamber, Jeffery C.; Doyley, Marvin M.; Leach, Martin O.

1997-04-01

Elasticity imaging (EI) has recently been proposed as a technique for imaging the mechanical properties of soft tissue. However, dynamic features, known as compressibility and mobility, are already employed to distinguish between different tissue types in ultrasound breast examination. This method, which involves the subjective interpretation of tissue motion seen in real-time B-mode images during palpation, is hereafter referred to as differential motion imaging (DMI). The purpose of this study was to develop the methodology required to perform a series of perception experiments to measure elastic lesion detectability by means of DMI and to obtain preliminary results for elastic contrast thresholds for different lesion sizes. Simulated sequences of real-time B-scans of tissue moving in response to an applied force were generated. A two-alternative forced choice (2-AFC) experiment was conducted and the measured contrast thresholds were compared with published results for lesions detected by EI. Although the trained observer was found to be quite skilled at the task of differential motion perception, it would appear that lesion detectability is improved when motion information is detected by computer processing and converted to gray scale before presentation to the observer. In particular, for lesions containing fewer than eight speckle cells, a signal detection rate of 100% could not be achieved even when the elastic contrast was very high.

Development of a real-time quantitative assay for detection of Epstein-Barr virus

NARCIS (Netherlands)

H.G.M. Niesters (Bert); E. Fries; K.C. Wolthers (Katja); A.D.M.E. Osterhaus (Albert); J.J. Cornelissen (Jan); J.W.J. van Esser (Joost)

2000-01-01

textabstractWith the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and

Quantitative studies of the distribution pattern for Salmonella Enteritidis in the internal organs of chicken after oral challenge by a real-time PCR.

Science.gov (United States)

He, G Z; Tian, W Y; Qian, N; Cheng, A C; Deng, S X

2010-12-01

This research was undertaken to identify and understand the regular distribution pattern for Salmonella Enteritidis (S. enteritidis) in the internal organs of chicken after oral challenge over a 3 wk period. We used a real-time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) to detect genomic DNA of S. enteritidis in the blood and the internal organs, including heart, liver, spleen, kidney, pancreas, and gallbladder, from chicken after oral challenge at different time points. The results showed that the spleen was positive at 12 h post inoculation (PI), and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, pancrea was positive at 20 h PI, and the final organ to show a positive results were the kidney and gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24 h-36 h PI, with the liver and spleen containing high concentrations of S. enteritidis, whereas the blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 12 d PI in the gallbladder, 2 wk for the liver, and 3 wk for the spleen without causing apparent symptoms. The results showed that the liver and spleen may be the primary sites for S. enteritidis setting itself up as a commensa over a long time after oral challenge. Interestingly, it may be the first time reported that the gallbladder is a site of carriage for S. enteritidis over a 12 d period. This study will help to understand the mechanisms of action of S. enteritidis infection in vivo.

Process algebra with timing : real time and discrete time

NARCIS (Netherlands)

Baeten, J.C.M.; Middelburg, C.A.; Bergstra, J.A.; Ponse, A.J.; Smolka, S.A.

2001-01-01

We present real time and discrete time versions of ACP with absolute timing and relative timing. The starting-point is a new real time version with absolute timing, called ACPsat, featuring urgent actions and a delay operator. The discrete time versions are conservative extensions of the discrete

Process algebra with timing: Real time and discrete time

NARCIS (Netherlands)

Baeten, J.C.M.; Middelburg, C.A.

1999-01-01

We present real time and discrete time versions of ACP with absolute timing and relative timing. The startingpoint is a new real time version with absolute timing, called ACPsat , featuring urgent actions and a delay operator. The discrete time versions are conservative extensions of the discrete

Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

Science.gov (United States)

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-01

Background Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. Methods The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. Conclusions The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. Significance The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food. PMID:21283808

Detection of polyoma virus in brain tissue of patients with progressive multifocal leukoencephalopathy by real-time PCR and pyrosequencing.

Science.gov (United States)

Beck, Rose C; Kohn, Debra J; Tuohy, Marion J; Prayson, Richard A; Yen-Lieberman, Belinda; Procop, Gary W

2004-03-01

We evaluated 2 methods, a LightCycler PCR assay and pyrosequencing for the detection of the JC polyoma virus (JCV) in fixed brain tissue of 10 patients with and 3 control patients without progressive multifocal leukoencephalopathy (PML). Nucleic acid extraction was performed after deparaffinization and proteinase K digestion. The LightCycler assay differentiates the BK virus (BKV), JCV, and SV40 using melt curve analysis. Conventional PCR was used with the same primers to generate products for pyrosequencing. Two sequencing primers were used that differentiate the polyoma viruses. Seven of 11 biopsies (1 patient had 2 biopsies) with PML were positive for JCV by real-time PCR and/or PCR/pyrosequencing. Three of 4 remaining biopsies were positive by real-time PCR but had melting points between JCV and SV40. The 4 specimens that were negative or atypical by LightCycler PCR were positive by traditional PCR, but 1 had an amplicon of lower molecular weight by gel electrophoresis. These were shown to represent JCV by at least 1 of the 2 pyrosequencing primers. The biopsies from patients without PML were PCR negative. Both the LightCycler and pyrosequencing assays are useful for confirming JCV in brain biopsies from patients with PML, but variant JCVs may require supplementary methods to confirm JCV infection.

MTF analysis of the near-real time neutron radiography facility at MURR

International Nuclear Information System (INIS)

Lindsay, J.T.; Alger, D.M.; Bull, S.R.; Miller, W.H.

1983-01-01

Several neutron radiography systems designed to view transient processes on a real-time basis have been developed. With the advent of these different real-time systems comes the necessity to develop a means to quantitatively evaluate and compare these systems. A suitable method for measuring the resolution capabilities of the image-forming system is the determination of the modulation transfer function (MTF). The MTF is a measure of an imaging system's ability to reproduce the spatial frequencies present in an image. The system in use at the University of Missouri Research Reactor is described. (Auth.)

Estimating marginal properties of quantitative real-time PCR data using nonlinear mixed models

DEFF Research Database (Denmark)

Gerhard, Daniel; Bremer, Melanie; Ritz, Christian

2014-01-01

A unified modeling framework based on a set of nonlinear mixed models is proposed for flexible modeling of gene expression in real-time PCR experiments. Focus is on estimating the marginal or population-based derived parameters: cycle thresholds and ΔΔc(t), but retaining the conditional mixed mod...

Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.).

Science.gov (United States)

Galeano, Esteban; Vasconcelos, Tarcísio Sales; Ramiro, Daniel Alves; De Martin, Valentina de Fátima; Carrer, Helaine

2014-07-22

Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. This study proposes a first broad

Real-time radiography

International Nuclear Information System (INIS)

Bossi, R.H.; Oien, C.T.

1981-01-01

Real-time radiography is used for imaging both dynamic events and static objects. Fluorescent screens play an important role in converting radiation to light, which is then observed directly or intensified and detected. The radiographic parameters for real-time radiography are similar to conventional film radiography with special emphasis on statistics and magnification. Direct-viewing fluoroscopy uses the human eye as a detector of fluorescent screen light or the light from an intensifier. Remote-viewing systems replace the human observer with a television camera. The remote-viewing systems have many advantages over the direct-viewing conditions such as safety, image enhancement, and the capability to produce permanent records. This report reviews real-time imaging system parameters and components

Real-Time PCR (qPCR) Primer Design Using Free Online Software

Science.gov (United States)

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

Quantification of bacteria adherent to gastrointestinal mucosa by real-time PCR

NARCIS (Netherlands)

Huijsdens, Xander W.; Linskens, Ronald K.; Mak, Mariëtte; Meuwissen, Stephan G. M.; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

2002-01-01

The use of real-time quantitative PCR (5' nuclease PCR assay) as a tool to study the gastrointestinal microflora that adheres to the colonic mucosa was evaluated. We developed primers and probes based on the 16S ribosomal DNA gene sequences for the detection of Escherichia coli and Bacteroides

Real-time vision systems

Energy Technology Data Exchange (ETDEWEB)

Johnson, R.; Hernandez, J.E.; Lu, Shin-yee [Lawrence Livermore National Lab., CA (United States)

1994-11-15

Many industrial and defence applications require an ability to make instantaneous decisions based on sensor input of a time varying process. Such systems are referred to as `real-time systems` because they process and act on data as it occurs in time. When a vision sensor is used in a real-time system, the processing demands can be quite substantial, with typical data rates of 10-20 million samples per second. A real-time Machine Vision Laboratory (MVL) was established in FY94 to extend our years of experience in developing computer vision algorithms to include the development and implementation of real-time vision systems. The laboratory is equipped with a variety of hardware components, including Datacube image acquisition and processing boards, a Sun workstation, and several different types of CCD cameras, including monochrome and color area cameras and analog and digital line-scan cameras. The equipment is reconfigurable for prototyping different applications. This facility has been used to support several programs at LLNL, including O Division`s Peacemaker and Deadeye Projects as well as the CRADA with the U.S. Textile Industry, CAFE (Computer Aided Fabric Inspection). To date, we have successfully demonstrated several real-time applications: bullet tracking, stereo tracking and ranging, and web inspection. This work has been documented in the ongoing development of a real-time software library.

Comparison of Abbott and Da-an real-time PCR for quantitating serum HBV DNA.

Science.gov (United States)

Qiu, Ning; Li, Rui; Yu, Jian-Guo; Yang, Wen; Zhang, Wei; An, Yong; Li, Tong; Liu, Xue-En; Zhuang, Hui

2014-09-07

To compare the performance of the Da-an real-time hepatitis B virus (HBV) DNA assay and Abbott RealTime HBV assay. HBV DNA standards as well as a total of 180 clinical serum samples from patients with chronic hepatitis B were measured using the Abbott and Da-an real-time polymerase chain reaction (PCR) assays. Correlation and Bland-Altman plot analysis was used to compare the performance of the Abbott and Da-an assays. The HBV DNA levels were logarithmically transformed for analysis. All statistical analyses were performed using SPSS for Windows version 18.0. The correlation between the two assays was analyzed by Pearson's correlation and linear regression. The Bland-Altman plots were used for the analysis of agreement between the two assays. A P value of Da-an assay were significantly correlated with the expected values of HBV DNA standards (r = 0.999, for Abbott; r = 0.987, for Da-an, P Da-an assay. Moreover, HBV DNA levels measured by the Abbott assay were significantly higher than those of the Da-an assay (6.23 ± 1.76 log IU/mL vs 5.46 ± 1.55 log IU/mL, P Da-an assay presented lower sensitivity and a narrower linear range as compared to the Abbott assay, suggesting the need to be improved.

Digital video timing analyzer for the evaluation of PC-based real-time simulation systems

Science.gov (United States)

Jones, Shawn R.; Crosby, Jay L.; Terry, John E., Jr.

2009-05-01

Due to the rapid acceleration in technology and the drop in costs, the use of commercial off-the-shelf (COTS) PC-based hardware and software components for digital and hardware-in-the-loop (HWIL) simulations has increased. However, the increase in PC-based components creates new challenges for HWIL test facilities such as cost-effective hardware and software selection, system configuration and integration, performance testing, and simulation verification/validation. This paper will discuss how the Digital Video Timing Analyzer (DiViTA) installed in the Aviation and Missile Research, Development and Engineering Center (AMRDEC) provides quantitative characterization data for PC-based real-time scene generation systems. An overview of the DiViTA is provided followed by details on measurement techniques, applications, and real-world examples of system benefits.

Quantitative analysis of myocardial tissue with digital autofluorescence microscopy

DEFF Research Database (Denmark)

Jensen, Thomas; Holten-Rossing, Henrik; Svendsen, Ida M H

2016-01-01

to that of hematoxylin and eosin staining in conventional pathology. This study presents an automated fluorescence-based microscopy approach providing highly detailed morphological data from unstained microsections. This data may provide a basic histological starting point from which further digital analysis including...... staining may benefit. METHODS: This study explores the inherent tissue fluorescence, also known as autofluorescence, as a mean to quantitate cardiac tissue components in histological microsections. Data acquisition using a commercially available whole slide scanner and an image-based quantitation algorithm......BACKGROUND: The opportunity offered by whole slide scanners of automated histological analysis implies an ever increasing importance of digital pathology. To go beyond the importance of conventional pathology, however, digital pathology may need a basic histological starting point similar...

Memory controllers for real-time embedded systems predictable and composable real-time systems

CERN Document Server

Akesson, Benny

2012-01-01

  Verification of real-time requirements in systems-on-chip becomes more complex as more applications are integrated. Predictable and composable systems can manage the increasing complexity using formal verification and simulation.  This book explains the concepts of predictability and composability and shows how to apply them to the design and analysis of a memory controller, which is a key component in any real-time system. This book is generally intended for readers interested in Systems-on-Chips with real-time applications.   It is especially well-suited for readers looking to use SDRAM memories in systems with hard or firm real-time requirements. There is a strong focus on real-time concepts, such as predictability and composability, as well as a brief discussion about memory controller architectures for high-performance computing. Readers will learn step-by-step how to go from an unpredictable SDRAM memory, offering highly variable bandwidth and latency, to a predictable and composable shared memory...

Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture.

Science.gov (United States)

de Wit, C; Fautz, C; Xu, Y

2000-09-01

Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1x10(6) particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3-6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5'-->3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600 particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell

A quantitative method to track protein translocation between intracellular compartments in real-time in live cells using weighted local variance image analysis.

Directory of Open Access Journals (Sweden)

Guillaume Calmettes

Full Text Available The genetic expression of cloned fluorescent proteins coupled to time-lapse fluorescence microscopy has opened the door to the direct visualization of a wide range of molecular interactions in living cells. In particular, the dynamic translocation of proteins can now be explored in real time at the single-cell level. Here we propose a reliable, easy-to-implement, quantitative image processing method to assess protein translocation in living cells based on the computation of spatial variance maps of time-lapse images. The method is first illustrated and validated on simulated images of a fluorescently-labeled protein translocating from mitochondria to cytoplasm, and then applied to experimental data obtained with fluorescently-labeled hexokinase 2 in different cell types imaged by regular or confocal microscopy. The method was found to be robust with respect to cell morphology changes and mitochondrial dynamics (fusion, fission, movement during the time-lapse imaging. Its ease of implementation should facilitate its application to a broad spectrum of time-lapse imaging studies.

A framework for predicting three-dimensional prostate deformation in real time

NARCIS (Netherlands)

Jahya, Alex; Herink, Mark; Misra, Sarthak

2013-01-01

Background Surgical simulation systems can be used to estimate soft tissue deformation during pre- and intra-operative planning. Such systems require a model that can accurately predict the deformation in real time. In this study, we present a back-propagation neural network for predicting

GPU-accelerated ray-tracing for real-time treatment planning

International Nuclear Information System (INIS)

Heinrich, H; Ziegenhein, P; Kamerling, C P; Oelfke, U; Froening, H

2014-01-01

Dose calculation methods in radiotherapy treatment planning require the radiological depth information of the voxels that represent the patient volume to correct for tissue inhomogeneities. This information is acquired by time consuming ray-tracing-based calculations. For treatment planning scenarios with changing geometries and real-time constraints this is a severe bottleneck. We implemented an algorithm for the graphics processing unit (GPU) which implements a ray-matrix approach to reduce the number of rays to trace. Furthermore, we investigated the impact of different strategies of accessing memory in kernel implementations as well as strategies for rapid data transfers between main memory and memory of the graphics device. Our study included the overlapping of computations and memory transfers to reduce the overall runtime using Hyper-Q. We tested our approach on a prostate case (9 beams, coplanar). The measured execution times for a complete ray-tracing range from 28 msec for the computations on the GPU to 99 msec when considering data transfers to and from the graphics device. Our GPU-based algorithm performed the ray-tracing in real-time. The strategies efficiently reduce the time consumption of memory accesses and data transfer overhead. The achieved runtimes demonstrate the viability of this approach and allow improved real-time performance for dose calculation methods in clinical routine.

Development of a real-time quantitative assay for detection of Epstein-Barr virus

NARCIS (Netherlands)

Niesters, H. G.; van Esser, J.; Fries, E.; Wolthers, K. C.; Cornelissen, J.; Osterhaus, A. D.

2000-01-01

With the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and 10(7) copies of DNA per ml using

DEVELOPMENT OF REAL-TIME MULTIPLEX PCR FOR THE QUANTITATIVE DETERMINATION OF TREC'S AND KREC'S IN WHOLE BLOOD AND IN DRIED BLOOD SPOTS

Directory of Open Access Journals (Sweden)

M. A. Gordukova

2015-01-01

Full Text Available Primary immunodeficiencies (PID such as severe combined immunodeficiency (SCID and X-linked agammaglobulinemia are characterized by the lack of functional Tand B-cells, respectively. Without early diagnosis and prompt treatment children with PID suffer from severe infectious diseases, leading to their death or disability. Our purpose was developing of simple, inexpensive, high throughput technique based on the quantitative determination of TREC and KREC molecules by real-time PCR, and its validation in a group of children with a verified diagnosis of SCID and X-linked agammaglobulinemia.In this study, we developed and validated multiplex real-time PCR for the TREC’s and KREC’s quantitative analysis. We have shown that linear range of Ct changes depending on the concentrations of targets with a correlation coefficient R2 not worse than 0.98 was observed at concentrations from 109 to 5 × 104 copies per ml. The lowest amount of targets reliably detected in a reaction volume was 10 TREC’s copies, 5 KREC ‘s copies and 5 copies of internal control (IL17RA. We determined the age-depended reference values of TRECs and KRECs in whole blood in 29 boys and 27 girls with normal immunological parameters. The normal cut-offs for TRECs and KRECs were defined in dry blood spots depending on the method of extraction.The proposed method showed 100% diagnostic sensitivity and specificity in the studied group. The method can be proposed as a screening tool for the diagnosis of SCID and X-linked agammaglobulinemia both in whole blood and in the dry blood spots. The further investigation is required with larger number of samples. 

Real-time individualized training vectors for experiential learning.

Energy Technology Data Exchange (ETDEWEB)

Willis, Matt; Tucker, Eilish Marie; Raybourn, Elaine Marie; Glickman, Matthew R.; Fabian, Nathan

2011-01-01

Military training utilizing serious games or virtual worlds potentially generate data that can be mined to better understand how trainees learn in experiential exercises. Few data mining approaches for deployed military training games exist. Opportunities exist to collect and analyze these data, as well as to construct a full-history learner model. Outcomes discussed in the present document include results from a quasi-experimental research study on military game-based experiential learning, the deployment of an online game for training evidence collection, and results from a proof-of-concept pilot study on the development of individualized training vectors. This Lab Directed Research & Development (LDRD) project leveraged products within projects, such as Titan (Network Grand Challenge), Real-Time Feedback and Evaluation System, (America's Army Adaptive Thinking and Leadership, DARWARS Ambush! NK), and Dynamic Bayesian Networks to investigate whether machine learning capabilities could perform real-time, in-game similarity vectors of learner performance, toward adaptation of content delivery, and quantitative measurement of experiential learning.

Visually estimated ejection fraction by two dimensional and triplane echocardiography is closely correlated with quantitative ejection fraction by real-time three dimensional echocardiography.

Science.gov (United States)

Shahgaldi, Kambiz; Gudmundsson, Petri; Manouras, Aristomenis; Brodin, Lars-Ake; Winter, Reidar

2009-08-25

Visual assessment of left ventricular ejection fraction (LVEF) is often used in clinical routine despite general recommendations to use quantitative biplane Simpsons (BPS) measurements. Even thou quantitative methods are well validated and from many reasons preferable, the feasibility of visual assessment (eyeballing) is superior. There is to date only sparse data comparing visual EF assessment in comparison to quantitative methods available. The aim of this study was to compare visual EF assessment by two-dimensional echocardiography (2DE) and triplane echocardiography (TPE) using quantitative real-time three-dimensional echocardiography (RT3DE) as the reference method. Thirty patients were enrolled in the study. Eyeballing EF was assessed using apical 4-and 2 chamber views and TP mode by two experienced readers blinded to all clinical data. The measurements were compared to quantitative RT3DE. There were an excellent correlation between eyeballing EF by 2D and TP vs 3DE (r = 0.91 and 0.95 respectively) without any significant bias (-0.5 +/- 3.7% and -0.2 +/- 2.9% respectively). Intraobserver variability was 3.8% for eyeballing 2DE, 3.2% for eyeballing TP and 2.3% for quantitative 3D-EF. Interobserver variability was 7.5% for eyeballing 2D and 8.4% for eyeballing TP. Visual estimation of LVEF both using 2D and TP by an experienced reader correlates well with quantitative EF determined by RT3DE. There is an apparent trend towards a smaller variability using TP in comparison to 2D, this was however not statistically significant.

Real-time zymography and reverse zymography: a method for detecting activities of matrix metalloproteinases and their inhibitors using FITC-labeled collagen and casein as substrates.

Science.gov (United States)

Hattori, Shunji; Fujisaki, Hitomi; Kiriyama, Tomomi; Yokoyama, Tsukao; Irie, Shinkichi

2002-02-01

Zymography and reverse zymography are widely used techniques for identifying the proteolytic activity of enzymes and the presence of protease inhibitors in polyacrylamide gels. In the current studies, we utilized a fluorescein-isothiocyanate-labeled substrate to develop novel zymographic and reverse zymographic methods for detecting matrix metalloproteinases and tissue inhibitors of the metalloproteinases, respectively. Using a transilluminator, the results can be observed visually without stopping the enzymatic reaction. For this reason, we have named these methods real-time zymography and real-time reverse zymography. These methods have the following advantages compared with conventional protocols: (1) because the reaction can be repeatedly monitored on the polyacrylamide gels, optimization of the incubation time can be achieved without preliminary analyses; (2) higher sensitivity is achieved with a lower amount of substrate than with conventional methods; (3) a semi-quantitative analysis of matrix metalloproteinases is possible. An additional advantage of the real-time reverse zymography is that, because the fluorescence detection is specific for substrate digestion, the inhibitor bands can be easily distinguished from contaminating proteins.

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Directory of Open Access Journals (Sweden)

Peng Xia

2009-01-01

Full Text Available Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf mitochondrial (mtDNA and nuclear (nDNA DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

Essays in real-time forecasting

OpenAIRE

Liebermann, Joelle

2012-01-01

This thesis contains three essays in the field of real-time econometrics, and more particularlyforecasting.The issue of using data as available in real-time to forecasters, policymakers or financialmarkets is an important one which has only recently been taken on board in the empiricalliterature. Data available and used in real-time are preliminary and differ from ex-postrevised data, and given that data revisions may be quite substantial, the use of latestavailable instead of real-time can s...

Using Non-Invasive Multi-Spectral Imaging to Quantitatively Assess Tissue Vasculature

Energy Technology Data Exchange (ETDEWEB)

Vogel, A; Chernomordik, V; Riley, J; Hassan, M; Amyot, F; Dasgeb, B; Demos, S G; Pursley, R; Little, R; Yarchoan, R; Tao, Y; Gandjbakhche, A H

2007-10-04

This research describes a non-invasive, non-contact method used to quantitatively analyze the functional characteristics of tissue. Multi-spectral images collected at several near-infrared wavelengths are input into a mathematical optical skin model that considers the contributions from different analytes in the epidermis and dermis skin layers. Through a reconstruction algorithm, we can quantify the percent of blood in a given area of tissue and the fraction of that blood that is oxygenated. Imaging normal tissue confirms previously reported values for the percent of blood in tissue and the percent of blood that is oxygenated in tissue and surrounding vasculature, for the normal state and when ischemia is induced. This methodology has been applied to assess vascular Kaposi's sarcoma lesions and the surrounding tissue before and during experimental therapies. The multi-spectral imaging technique has been combined with laser Doppler imaging to gain additional information. Results indicate that these techniques are able to provide quantitative and functional information about tissue changes during experimental drug therapy and investigate progression of disease before changes are visibly apparent, suggesting a potential for them to be used as complementary imaging techniques to clinical assessment.

Platform for Automated Real-Time High Performance Analytics on Medical Image Data.

Science.gov (United States)

Allen, William J; Gabr, Refaat E; Tefera, Getaneh B; Pednekar, Amol S; Vaughn, Matthew W; Narayana, Ponnada A

2018-03-01

Biomedical data are quickly growing in volume and in variety, providing clinicians an opportunity for better clinical decision support. Here, we demonstrate a robust platform that uses software automation and high performance computing (HPC) resources to achieve real-time analytics of clinical data, specifically magnetic resonance imaging (MRI) data. We used the Agave application programming interface to facilitate communication, data transfer, and job control between an MRI scanner and an off-site HPC resource. In this use case, Agave executed the graphical pipeline tool GRAphical Pipeline Environment (GRAPE) to perform automated, real-time, quantitative analysis of MRI scans. Same-session image processing will open the door for adaptive scanning and real-time quality control, potentially accelerating the discovery of pathologies and minimizing patient callbacks. We envision this platform can be adapted to other medical instruments, HPC resources, and analytics tools.

Real-time quantification of subcellular H2O2 and glutathione redox potential in living cardiovascular tissues.

Science.gov (United States)

Panieri, Emiliano; Millia, Carlo; Santoro, Massimo M

2017-08-01

Detecting and measuring the dynamic redox events that occur in vivo is a prerequisite for understanding the impact of oxidants and redox events in normal and pathological conditions. These aspects are particularly relevant in cardiovascular tissues wherein alterations of the redox balance are associated with stroke, aging, and pharmacological intervention. An ambiguous aspect of redox biology is how redox events occur in subcellular organelles including mitochondria, and nuclei. Genetically-encoded Rogfp2 fluorescent probes have become powerful tools for real-time detection of redox events. These probes detect hydrogen peroxide (H 2 O 2 ) levels and glutathione redox potential (E GSH ), both with high spatiotemporal resolution. By generating novel transgenic (Tg) zebrafish lines that express compartment-specific Rogfp2-Orp1 and Grx1-Rogfp2 sensors we analyzed cytosolic, mitochondrial, and the nuclear redox state of endothelial cells and cardiomyocytes of living zebrafish embryos. We provide evidence for the usefulness of these Tg lines for pharmacological compounds screening by addressing the blocking of pentose phosphate pathways (PPP) and glutathione synthesis, thus altering subcellular redox state in vivo. Rogfp2-based transgenic zebrafish lines represent valuable tools to characterize the impact of redox changes in living tissues and offer new opportunities for studying metabolic driven antioxidant response in biomedical research. Copyright © 2017 Elsevier Inc. All rights reserved.

Ultrasound contrast agent imaging: Real-time imaging of the superharmonics

Energy Technology Data Exchange (ETDEWEB)

Peruzzini, D.; Viti, J. [MSD lab, Department of Information Engineering, Univ of Florence, Via S.Marta, 3, 50139 Firenze (Italy); Erasmus MC, ’s-Gravendijkwal 230, Faculty Building, Ee 2302, 3015 CE Rotterdam (Netherlands); Tortoli, P. [MSD lab, Department of Information Engineering, Univ of Florence, Via S.Marta, 3, 50139 Firenze (Italy); Verweij, M. D. [Acoustical Wavefield Imaging, ImPhys, Delft Univ Technology, van der Waalsweg 8, 2628 CH Delft (Netherlands); Jong, N. de; Vos, H. J., E-mail: h.vos@erasmusmc.nl [Erasmus MC, ’s-Gravendijkwal 230, Faculty Building, Ee 2302, 3015 CE Rotterdam (Netherlands); Acoustical Wavefield Imaging, ImPhys, Delft Univ Technology, van der Waalsweg 8, 2628 CH Delft (Netherlands)

2015-10-28

Currently, in medical ultrasound contrast agent (UCA) imaging the second harmonic scattering of the microbubbles is regularly used. This scattering is in competition with the signal that is caused by nonlinear wave propagation in tissue. It was reported that UCA imaging based on the third or higher harmonics, i.e. “superharmonic” imaging, shows better contrast. However, the superharmonic scattering has a lower signal level compared to e.g. second harmonic signals. This study investigates the contrast-to-tissue ratio (CTR) and signal to noise ratio (SNR) of superharmonic UCA scattering in a tissue/vessel mimicking phantom using a real-time clinical scanner. Numerical simulations were performed to estimate the level of harmonics generated by the microbubbles. Data were acquired with a custom built dual-frequency cardiac phased array probe. Fundamental real-time images were produced while beam formed radiofrequency (RF) data was stored for further offline processing. The phantom consisted of a cavity filled with UCA surrounded by tissue mimicking material. The acoustic pressure in the cavity of the phantom was 110 kPa (MI = 0.11) ensuring non-destructivity of UCA. After processing of the acquired data from the phantom, the UCA-filled cavity could be clearly observed in the images, while tissue signals were suppressed at or below the noise floor. The measured CTR values were 36 dB, >38 dB, and >32 dB, for the second, third, and fourth harmonic respectively, which were in agreement with those reported earlier for preliminary contrast superharmonic imaging. The single frame SNR values (in which ‘signal’ denotes the signal level from the UCA area) were 23 dB, 18 dB, and 11 dB, respectively. This indicates that noise, and not the tissue signal, is the limiting factor for the UCA detection when using the superharmonics in nondestructive mode.

Ovation Prime Real-Time

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — The Ovation Prime Real-Time (OPRT) product is a real-time forecast and nowcast model of auroral power and is an operational implementation of the work by Newell et...

Evaluation of Housekeeping Genes for Quantitative Real-Time PCR Analysis of Bradysia odoriphaga (Diptera: Sciaridae

Directory of Open Access Journals (Sweden)

Caihua Shi

2016-07-01

Full Text Available The soil insect Bradysia odoriphaga (Diptera: Sciaridae causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR. This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga.

Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children.

Science.gov (United States)

Pitetti, Raymond D; Laus, Stella; Wadowsky, Robert M

2003-08-01

Epstein-Barr virus (EBV) infectious mononucleosis is often diagnosed based on characteristic clinical features and either a positive heterophil antibody test or serology, both of which can be unreliable in young children. Real time quantitative PCR assays that measure EBV DNA load in serum or plasma are highly sensitive in young children, but serum and plasma contain inhibitors of PCR which must be removed by DNA extraction techniques. A real time TaqMan PCR assay was designed and evaluated for simultaneously measuring EBV DNA load and validating the removal of PCR inhibitors from serum samples. A serum sample was available from patients classified serologically as primary EBV infection (n = 28), EBV-seronegative (n = 25) and EBV-seropositive (n = 26). Patients were classified as having EBV infectious mononucleosis if they had specified clinical findings and > or =10% atypical lymphocytes in peripheral blood or had a positive Monospot test result. DNA was purified by a spin column method and tested in PCR reactions with primers for EBV DNA polymerase gene and internal control targets. Amplification of the two PCR products was measured in real time with separate TaqMan DNA probes labeled with various fluorescent reporters. The mean age of study patients was 9 years, 4 months. Twenty-one (75%) of the patients in the primary EBV infection group, one (4%) of the seronegatives and none of the seropositives had detectable EBV DNA. Within the primary infection group, those with detectable virus were more likely than those without detectable virus to have evidence of lymphadenopathy (14 of 16 vs.1 of 5; P = 0.011), higher mean atypical (11.7 vs.0.9%; P = 0.002) and absolute atypical (1.5 vs.0.1 x 109/l; P = 0.004) lymphocyte count, higher mean absolute lymphocyte count (4.7 vs.2.3 x 109/l; P = 0.026) and higher mean aspartate aminotransferase value (119.8 vs.37.3 IU/l; P = 0.036). Ten patients, all in the primary infection group, had EBV infectious mononucleosis, and all

In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues

Science.gov (United States)

Biris, Alexandru S.; Galanzha, Ekaterina I.; Li, Zhongrui; Mahmood, Meena; Xu, Yang; Zharov, Vladimir P.

2009-03-01

Nanoparticles are intensively being explored as contrast agents for medical diagnostics and therapies using various optical methods. We present the first demonstration of the use of time-resolved Raman spectroscopy for in vivo real-time detection of circulating carbon nanotubes (CNTs) or cancer cells labeled with CNTs in the lymph, blood, and tissues of live animals with fast spectral acquisition times of down to few milliseconds. After intravenously administering CNTs in the tail vein of the rat, this technique provides the ability to detect the circulation of CNTs in the blood microvessels of the intact rat ear. The capability of Raman spectroscopy is also demonstrated to monitor, identify, and image the CNTs during their transportation by lymphatics in the rat ear and mesentery. The strong and specific Raman scattering properties of CNTs make it possible to detect in vitro and in vivo single cancer cells (HeLa) tagged with CNTs. In vivo Raman flow cytometry opens a new avenue for multiparameter analysis of circulating nanoparticles with strong Raman scattering properties and their pharmokinetics in blood and lymph systems. Moreover, this technology has the potential for molecular detection and identification of circulating tumor cells, and infections labeled with CNTs.

VERSE - Virtual Equivalent Real-time Simulation

Science.gov (United States)

Zheng, Yang; Martin, Bryan J.; Villaume, Nathaniel

2005-01-01

Distributed real-time simulations provide important timing validation and hardware in the- loop results for the spacecraft flight software development cycle. Occasionally, the need for higher fidelity modeling and more comprehensive debugging capabilities - combined with a limited amount of computational resources - calls for a non real-time simulation environment that mimics the real-time environment. By creating a non real-time environment that accommodates simulations and flight software designed for a multi-CPU real-time system, we can save development time, cut mission costs, and reduce the likelihood of errors. This paper presents such a solution: Virtual Equivalent Real-time Simulation Environment (VERSE). VERSE turns the real-time operating system RTAI (Real-time Application Interface) into an event driven simulator that runs in virtual real time. Designed to keep the original RTAI architecture as intact as possible, and therefore inheriting RTAI's many capabilities, VERSE was implemented with remarkably little change to the RTAI source code. This small footprint together with use of the same API allows users to easily run the same application in both real-time and virtual time environments. VERSE has been used to build a workstation testbed for NASA's Space Interferometry Mission (SIM PlanetQuest) instrument flight software. With its flexible simulation controls and inexpensive setup and replication costs, VERSE will become an invaluable tool in future mission development.

A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR

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Stewart Don

2008-05-01

Full Text Available Abstract Background Based upon defining a common reference point, current real-time quantitative PCR technologies compare relative differences in amplification profile position. As such, absolute quantification requires construction of target-specific standard curves that are highly resource intensive and prone to introducing quantitative errors. Sigmoidal modeling using nonlinear regression has previously demonstrated that absolute quantification can be accomplished without standard curves; however, quantitative errors caused by distortions within the plateau phase have impeded effective implementation of this alternative approach. Results Recognition that amplification rate is linearly correlated to amplicon quantity led to the derivation of two sigmoid functions that allow target quantification via linear regression analysis. In addition to circumventing quantitative errors produced by plateau distortions, this approach allows the amplification efficiency within individual amplification reactions to be determined. Absolute quantification is accomplished by first converting individual fluorescence readings into target quantity expressed in fluorescence units, followed by conversion into the number of target molecules via optical calibration. Founded upon expressing reaction fluorescence in relation to amplicon DNA mass, a seminal element of this study was to implement optical calibration using lambda gDNA as a universal quantitative standard. Not only does this eliminate the need to prepare target-specific quantitative standards, it relegates establishment of quantitative scale to a single, highly defined entity. The quantitative competency of this approach was assessed by exploiting "limiting dilution assay" for absolute quantification, which provided an independent gold standard from which to verify quantitative accuracy. This yielded substantive corroborating evidence that absolute accuracies of ± 25% can be routinely achieved. Comparison

Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction.

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Petersen, M I; Alvarez, I; Trono, K G; Jaworski, J P

2018-04-11

The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

International Nuclear Information System (INIS)

Cicinnati, Vito R; Shen, Qingli; Sotiropoulos, Georgios C; Radtke, Arnold; Gerken, Guido; Beckebaum, Susanne

2008-01-01

Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented. Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

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Radtke Arnold

2008-11-01

Full Text Available Abstract Background Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR. The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC is presented. Methods Six genes, beta-2-microglobulin (B2M, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hydroxymethyl-bilane synthase (HMBS, hypoxanthine phosphoribosyl-transferase 1 (HPRT1, succinate dehydrogenase complex, subunit A (SDHA and ubiquitin C (UBC, with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. Results HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Conclusion Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

Science.gov (United States)

Cicinnati, Vito R; Shen, Qingli; Sotiropoulos, Georgios C; Radtke, Arnold; Gerken, Guido; Beckebaum, Susanne

2008-01-01

Background Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented. Methods Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. Results HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Conclusion Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues

Tendency for interlaboratory precision in the GMO analysis method based on real-time PCR.

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Kodama, Takashi; Kurosawa, Yasunori; Kitta, Kazumi; Naito, Shigehiro

2010-01-01

The Horwitz curve estimates interlaboratory precision as a function only of concentration, and is frequently used as a method performance criterion in food analysis with chemical methods. The quantitative biochemical methods based on real-time PCR require an analogous criterion to progressively promote method validation. We analyzed the tendency of precision using a simplex real-time PCR technique in 53 collaborative studies of seven genetically modified (GM) crops. Reproducibility standard deviation (SR) and repeatability standard deviation (Sr) of the genetically modified organism (GMO) amount (%) was more or less independent of GM crops (i.e., maize, soybean, cotton, oilseed rape, potato, sugar beet, and rice) and evaluation procedure steps. Some studies evaluated whole steps consisting of DNA extraction and PCR quantitation, whereas others focused only on the PCR quantitation step by using DNA extraction solutions. Therefore, SR and Sr for GMO amount (%) are functions only of concentration similar to the Horwitz curve. We proposed S(R) = 0.1971C 0.8685 and S(r) = 0.1478C 0.8424, where C is the GMO amount (%). We also proposed a method performance index in GMO quantitative methods that is analogous to the Horwitz Ratio.

The quantification of spermatozoa by real-time quantitative PCR, spectrophotometry, and spermatophore cap size.

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Doyle, Jacqueline M; McCormick, Cory R; DeWoody, J Andrew

2011-01-01

Many animals, such as crustaceans, insects, and salamanders, package their sperm into spermatophores, and the number of spermatozoa contained in a spermatophore is relevant to studies of sexual selection and sperm competition. We used two molecular methods, real-time quantitative polymerase chain reaction (RT-qPCR) and spectrophotometry, to estimate sperm numbers from spermatophores. First, we designed gene-specific primers that produced a single amplicon in four species of ambystomatid salamanders. A standard curve generated from cloned amplicons revealed a strong positive relationship between template DNA quantity and cycle threshold, suggesting that RT-qPCR could be used to quantify sperm in a given sample. We then extracted DNA from multiple Ambystoma maculatum spermatophores, performed RT-qPCR on each sample, and estimated template copy numbers (i.e. sperm number) using the standard curve. Second, we used spectrophotometry to determine the number of sperm per spermatophore by measuring DNA concentration relative to the genome size. We documented a significant positive relationship between the estimates of sperm number based on RT-qPCR and those based on spectrophotometry. When these molecular estimates were compared to spermatophore cap size, which in principle could predict the number of sperm contained in the spermatophore, we also found a significant positive relationship between sperm number and spermatophore cap size. This linear model allows estimates of sperm number strictly from cap size, an approach which could greatly simplify the estimation of sperm number in future studies. These methods may help explain variation in fertilization success where sperm competition is mediated by sperm quantity. © 2010 Blackwell Publishing Ltd.

[Comparison of two different real-time PCR systems in postmortem diagnosis of tuberculosis in paraffin-embedded tissues].

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Yağmur, Gülhan; Albayrak, Nurhan; Daş, Taner; Yıldırım, Muzaffer; Ozgün, Ayşe; Büyük, Yalçın

2014-10-01

Tuberculosis (TB) is one of those infections with high morbidity and mortality in all around the world. Hundreds of people died from this disease without diagnosed or due to resistant strains in Turkey. Therefore, it is important to identify postmortem cases who have died from tuberculosis. Molecular methods have been widely used as well as conventional methods in the diagnosis of tuberculosis. The aim of this study was to compare the two different real-time polymerase chain reaction (Rt-PCR) system in the postmortem diagnosis of Mycobacterium tuberculosis infections in paraffin-embedded tissues. A total of 40 paraffin-embedded tissue samples [lung (n= 35), brain (n= 2), heart (n= 2), lymph node (n= 1)] in which histopathologic findings consistent with TB (necrotizing granulomatous inflammation, gelatinous caseous pneumonia, necrotic fibrous nodul) obtained from 37 autopsy cases (31 male, 6 female; age range: 25-85 yrs) were included in the study. Paraffin-embedded tissues were deparafinized with xylene and ethyl alcohol and then DNA isolation was done with QIAsymphony DSP Virus/Pathogen Midi kit in the QIAsymphony device. DNA amplification process was performed by Rt-PCR using the kit Artus® M. tuberculosis RG-PCR in the Rotor-Gene® Q device (Qiagen, Germany). Likewise, after deparafinization process, samples placed in the cartridge and isolation and Rt-PCR was performed by Xpert® MTB/RIF (Cepheid, USA) system, simultaneosly. Seventeen and 20 out of the 40 paraffin-embedded tissues yielded positive results with Qiagen and Xpert system, respectively. M.tuberculosis DNA was found positive in 13 (32.5%) and negative in 16 (40%) of the samples by both of the systems, exhibiting 72.5% (29/40) of concordance. On the other hand, seven (17.5%) samples that were positive with Xpert system yielded negative result with the Qiagen, while four (10%) samples that were positive with Qiagen yielded negative result with the Xpert system. Of the 20 positive cases detected with

Visually estimated ejection fraction by two dimensional and triplane echocardiography is closely correlated with quantitative ejection fraction by real-time three dimensional echocardiography

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Manouras Aristomenis

2009-08-01

Full Text Available Abstract Background Visual assessment of left ventricular ejection fraction (LVEF is often used in clinical routine despite general recommendations to use quantitative biplane Simpsons (BPS measurements. Even thou quantitative methods are well validated and from many reasons preferable, the feasibility of visual assessment (eyeballing is superior. There is to date only sparse data comparing visual EF assessment in comparison to quantitative methods available. The aim of this study was to compare visual EF assessment by two-dimensional echocardiography (2DE and triplane echocardiography (TPE using quantitative real-time three-dimensional echocardiography (RT3DE as the reference method. Methods Thirty patients were enrolled in the study. Eyeballing EF was assessed using apical 4-and 2 chamber views and TP mode by two experienced readers blinded to all clinical data. The measurements were compared to quantitative RT3DE. Results There were an excellent correlation between eyeballing EF by 2D and TP vs 3DE (r = 0.91 and 0.95 respectively without any significant bias (-0.5 ± 3.7% and -0.2 ± 2.9% respectively. Intraobserver variability was 3.8% for eyeballing 2DE, 3.2% for eyeballing TP and 2.3% for quantitative 3D-EF. Interobserver variability was 7.5% for eyeballing 2D and 8.4% for eyeballing TP. Conclusion Visual estimation of LVEF both using 2D and TP by an experienced reader correlates well with quantitative EF determined by RT3DE. There is an apparent trend towards a smaller variability using TP in comparison to 2D, this was however not statistically significant.

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.).

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Vera-Jimenez, N I; Pietretti, D; Wiegertjes, G F; Nielsen, M E

2013-05-01

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans. Copyright © 2013 Elsevier Ltd. All rights reserved.

ISTTOK real-time architecture

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Carvalho, Ivo S., E-mail: ivoc@ipfn.ist.utl.pt; Duarte, Paulo; Fernandes, Horácio; Valcárcel, Daniel F.; Carvalho, Pedro J.; Silva, Carlos; Duarte, André S.; Neto, André; Sousa, Jorge; Batista, António J.N.; Hekkert, Tiago; Carvalho, Bernardo B.

2014-03-15

Highlights: • All real-time diagnostics and actuators were integrated in the same control platform. • A 100 μs control cycle was achieved under the MARTe framework. • Time-windows based control with several event-driven control strategies implemented. • AC discharges with exception handling on iron core flux saturation. • An HTML discharge configuration was developed for configuring the MARTe system. - Abstract: The ISTTOK tokamak was upgraded with a plasma control system based on the Advanced Telecommunications Computing Architecture (ATCA) standard. This control system was designed to improve the discharge stability and to extend the operational space to the alternate plasma current (AC) discharges as part of the ISTTOK scientific program. In order to accomplish these objectives all ISTTOK diagnostics and actuators relevant for real-time operation were integrated in the control system. The control system was programmed in C++ over the Multi-threaded Application Real-Time executor (MARTe) which provides, among other features, a real-time scheduler, an interrupt handler, an intercommunications interface between code blocks and a clearly bounded interface with the external devices. As a complement to the MARTe framework, the BaseLib2 library provides the foundations for the data, code introspection and also a Hypertext Transfer Protocol (HTTP) server service. Taking advantage of the modular nature of MARTe, the algorithms of each diagnostic data processing, discharge timing, context switch, control and actuators output reference generation, run on well-defined blocks of code named Generic Application Module (GAM). This approach allows reusability of the code, simplified simulation, replacement or editing without changing the remaining GAMs. The ISTTOK control system GAMs run sequentially each 100 μs cycle on an Intel{sup ®} Q8200 4-core processor running at 2.33 GHz located in the ATCA crate. Two boards (inside the ATCA crate) with 32 analog

ISTTOK real-time architecture

International Nuclear Information System (INIS)

Carvalho, Ivo S.; Duarte, Paulo; Fernandes, Horácio; Valcárcel, Daniel F.; Carvalho, Pedro J.; Silva, Carlos; Duarte, André S.; Neto, André; Sousa, Jorge; Batista, António J.N.; Hekkert, Tiago; Carvalho, Bernardo B.

2014-01-01

Highlights: • All real-time diagnostics and actuators were integrated in the same control platform. • A 100 μs control cycle was achieved under the MARTe framework. • Time-windows based control with several event-driven control strategies implemented. • AC discharges with exception handling on iron core flux saturation. • An HTML discharge configuration was developed for configuring the MARTe system. - Abstract: The ISTTOK tokamak was upgraded with a plasma control system based on the Advanced Telecommunications Computing Architecture (ATCA) standard. This control system was designed to improve the discharge stability and to extend the operational space to the alternate plasma current (AC) discharges as part of the ISTTOK scientific program. In order to accomplish these objectives all ISTTOK diagnostics and actuators relevant for real-time operation were integrated in the control system. The control system was programmed in C++ over the Multi-threaded Application Real-Time executor (MARTe) which provides, among other features, a real-time scheduler, an interrupt handler, an intercommunications interface between code blocks and a clearly bounded interface with the external devices. As a complement to the MARTe framework, the BaseLib2 library provides the foundations for the data, code introspection and also a Hypertext Transfer Protocol (HTTP) server service. Taking advantage of the modular nature of MARTe, the algorithms of each diagnostic data processing, discharge timing, context switch, control and actuators output reference generation, run on well-defined blocks of code named Generic Application Module (GAM). This approach allows reusability of the code, simplified simulation, replacement or editing without changing the remaining GAMs. The ISTTOK control system GAMs run sequentially each 100 μs cycle on an Intel ® Q8200 4-core processor running at 2.33 GHz located in the ATCA crate. Two boards (inside the ATCA crate) with 32 analog

Effectiveness of quantitative real time PCR in long-term follow-up of chronic myeloid leukemia patients

International Nuclear Information System (INIS)

Savasoglu, K.; Berber, B.

2015-01-01

To determine the use of the Quantitative Real Time PCR (RQ-PCR) assay follow-up with Chronic Myeloid Leukemia (CML) patients. Study Design: Cross-sectional observational. Place and Duration of Study: Izmir Ataturk Education and Research Hospital, Izmir, Turkey, from 2009 to 2013. Methodology: Cytogenetic, FISH, RQ-PCR test results from 177 CML patients materials selected between 2009 - 2013 years was set up for comparison analysis. Statistical analysis was performed to compare between FISH, karyotype and RQ-PCR results of the patients. Karyotyping and FISH specificity and sensitivity rates determined by ROC analysis compared with RQ-PCR results. Chi-square test was used to compare test failure rates. Results:Sensitivity and specificity values were determined for karyotyping 17.6 - 98% (p=0.118, p > 0.05) and for FISH 22.5 - 96% (p=0.064, p > 0.05) respectively. FISH sensitivity was slightly higher than karyotyping but there was calculated a strong correlation between them (p < 0.001). RQ-PCR test failure rate did not correlate with other two tests (p > 0.05); however, karyotyping and FISH test failure rate was statistically significant (p < 0.001). Conclusion: Besides, the situation needed for karyotype analysis, RQ-PCR assay can be used alone in the follow-up of CML disease. (author)

Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

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Renata Stolf-Moreira

2011-01-01

Full Text Available The objective of this work was to validate, by quantitative PCR in real time (RT-qPCR, genes to be used as reference in studies of gene expression in soybean in drought-stressed trials. Four genes commonly used in soybean were evaluated: Gmβ-actin, GmGAPDH, GmLectin and GmRNAr18S. Total RNA was extracted from six samples: three from roots in a hydroponic system with different drought intensities (0, 25, 50, 75 and 100 minutes of water stress, and three from leaves of plants grown in sand with different soil moistures (15, 5 and 2.5% gravimetric humidity. The raw cycle threshold (Ct data were analyzed, and the efficiency of each primer was calculated for an overall analysis of the Ct range among the different samples. The GeNorm application was used to evaluate the best reference gene, according to its stability. The GmGAPDH was the least stable gene, with the highest mean values of expression stability (M, and the most stable genes, with the lowest M values, were the Gmβ-actin and GmRNAr18S, when both root and leaves samples were tested. These genes can be used in RT-qPCR as reference gene for expression analysis.

A novel semi-quantitative method for measuring tissue bleeding.

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Vukcevic, G; Volarevic, V; Raicevic, S; Tanaskovic, I; Milicic, B; Vulovic, T; Arsenijevic, S

2014-03-01

In this study, we describe a new semi-quantitative method for measuring the extent of bleeding in pathohistological tissue samples. To test our novel method, we recruited 120 female patients in their first trimester of pregnancy and divided them into three groups of 40. Group I was the control group, in which no dilation was applied. Group II was an experimental group, in which dilation was performed using classical mechanical dilators. Group III was also an experimental group, in which dilation was performed using a hydraulic dilator. Tissue samples were taken from the patients' cervical canals using a Novak's probe via energetic single-step curettage prior to any dilation in Group I and after dilation in Groups II and III. After the tissue samples were prepared, light microscopy was used to obtain microphotographs at 100x magnification. The surfaces affected by bleeding were measured in the microphotographs using the Autodesk AutoCAD 2009 program and its "polylines" function. The lines were used to mark the area around the entire sample (marked A) and to create "polyline" areas around each bleeding area on the sample (marked B). The percentage of the total area affected by bleeding was calculated using the formula: N = Bt x 100 / At where N is the percentage (%) of the tissue sample surface affected by bleeding, At (A total) is the sum of the surfaces of all of the tissue samples and Bt (B total) is the sum of all the surfaces affected by bleeding in all of the tissue samples. This novel semi-quantitative method utilizes the Autodesk AutoCAD 2009 program, which is simple to use and widely available, thereby offering a new, objective and precise approach to estimate the extent of bleeding in tissue samples.

Real time elastography - a non-invasive diagnostic method of small hepatocellular carcinoma in cirrhosis.

Science.gov (United States)

Gheorghe, Liana; Iacob, Speranta; Iacob, Razvan; Dumbrava, Mona; Becheanu, Gabriel; Herlea, Vlad; Gheorghe, Cristian; Lupescu, Ioana; Popescu, Irinel

2009-12-01

Small nodules (under 3 cm) detected on ultrasound (US) in cirrhotics represent the most challenging category for noninvasive diagnosis of hepatocellular carcinoma (HCC). To evaluate real-time sonoelastography as a noninvasive tool for the diagnosis of small HCC nodules in cirrhotic patients. 42 cirrhotic patients with 58 nodules (1-3 cm) were evaluated with real-time elastography (Hitachi EUB-6500); the mean intensity of colors red, blue, green were measured using a semi-quantitative method. Analysis of histograms for each color of the sonoelastography images was performed for quantifying the elasticity of nodule tissue in comparison with the cirrhotic liver tissue. AUROC curves were constructed to define the best cut-off points to distinguish malignant features of the nodules. Univariate and multivariate logistic regression analysis was performed. 595 sonoelastography images from 42 patients (25 men; 17 women) were analyzed. The mean age was 56.4 +/- 0.7 years and 69% patients were in Child-Pugh class A, 19% class B, 11% class C. For the mean intensity of green color AUROC=0.81, a cut-off value under 108.7 being diagnostic for HCC with a Sp=91.1%, Se=50%, PPV=92.1%, NPV=47.1%. Mean intensity of blue color proved to be an excellent diagnostic tool for HCC (AUROC=0.94); for a cut-off value greater than 128.9, Sp=92.2%, Se=78.9%, PPV=95.4%, NPV=68%. Independent predictive factors of HCC for a small nodule in cirrhotic patients were: blue color over 128.9 at sonoelastography and hypervascular appearance at Doppler US. US elastography is a promising method for the non-invasive diagnosis of early HCC. Blue color at elastography and hypervascular aspects are independent predictors of HCC.

A quantitative and non-contact technique to characterise microstructural variations of skin tissues during photo-damaging process based on Mueller matrix polarimetry.

Science.gov (United States)

Dong, Yang; He, Honghui; Sheng, Wei; Wu, Jian; Ma, Hui

2017-10-31

Skin tissue consists of collagen and elastic fibres, which are highly susceptible to damage when exposed to ultraviolet radiation (UVR), leading to skin aging and cancer. However, a lack of non-invasive detection methods makes determining the degree of UVR damage to skin in real time difficult. As one of the fundamental features of light, polarization can be used to develop imaging techniques capable of providing structural information about tissues. In particular, Mueller matrix polarimetry is suitable for detecting changes in collagen and elastic fibres. Here, we demonstrate a novel, quantitative, non-contact and in situ technique based on Mueller matrix polarimetry for monitoring the microstructural changes of skin tissues during UVR-induced photo-damaging. We measured the Mueller matrices of nude mouse skin samples, then analysed the transformed parameters to characterise microstructural changes during the skin photo-damaging and self-repairing processes. Comparisons between samples with and without the application of a sunscreen showed that the Mueller matrix-derived parameters are potential indicators for fibrous microstructure in skin tissues. Histological examination and Monte Carlo simulations confirmed the relationship between the Mueller matrix parameters and changes to fibrous structures. This technique paves the way for non-contact evaluation of skin structure in cosmetics and dermatological health.

A meshless EFG-based algorithm for 3D deformable modeling of soft tissue in real-time.

Science.gov (United States)

Abdi, Elahe; Farahmand, Farzam; Durali, Mohammad

2012-01-01

The meshless element-free Galerkin method was generalized and an algorithm was developed for 3D dynamic modeling of deformable bodies in real time. The efficacy of the algorithm was investigated in a 3D linear viscoelastic model of human spleen subjected to a time-varying compressive force exerted by a surgical grasper. The model remained stable in spite of the considerably large deformations occurred. There was a good agreement between the results and those of an equivalent finite element model. The computational cost, however, was much lower, enabling the proposed algorithm to be effectively used in real-time applications.

Observation of rat's colon polyps in real time by mini-endoscopy and raman spectroscopy

Science.gov (United States)

Andriana, Bibin Bintang; Mahardika, Anggara; Taketani, Akihiro; Sato, Hidetoshi

2018-02-01

Colorectal adenoma (CA) is a disease caused by various factors (such as genetic factors or environmental exposures). The appearance of colon polyp (CP) within colorectal might indicate the hint of CA development. Ball-lens hollow fiber Raman probe (BHRP) may has a high capability for detection of CA in living experimental animal and have already tested to rat's CP in this study, which was designed to collaborate between BHRP with mini-endoscopy to observe the biochemical alteration within normal colon tissue and rat's colon polyps in real time. BHRP and mini-endoscopy can distinguish the differences in their finger print spectra and make pictures the control and CP in the real time. At the first step, the real situation of normal colon and Rat's CP were washed by saline and observed with mini-endoscopy. BHRP was introduced to Dextran sodium sulphate (DSS)-induced Rat's CP to detect some of biochemical alteration. The main purpose of this study was to introduce mini-endoscopy to guide the BHRP for diagnosing of CP in real time and to compare it with spectra of normal colon (control group) in living rat. As the result, BHRP can provide the differences in band of control and CP group, which can inform that the biochemical of normal and CP has changed. As a major parameter to distinct normal and CP tissue were phosphatidylinositol, phosphodiester group, lipid, and collagen. Mini endoscopy and BHRP is very sensitive devices for diagnosing of CP in real time.

A Stereological Method for the Quantitative Evaluation of Cartilage Repair Tissue

Science.gov (United States)

Nyengaard, Jens Randel; Lind, Martin; Spector, Myron

2015-01-01

Objective To implement stereological principles to develop an easy applicable algorithm for unbiased and quantitative evaluation of cartilage repair. Design Design-unbiased sampling was performed by systematically sectioning the defect perpendicular to the joint surface in parallel planes providing 7 to 10 hematoxylin–eosin stained histological sections. Counting windows were systematically selected and converted into image files (40-50 per defect). The quantification was performed by two-step point counting: (1) calculation of defect volume and (2) quantitative analysis of tissue composition. Step 2 was performed by assigning each point to one of the following categories based on validated and easy distinguishable morphological characteristics: (1) hyaline cartilage (rounded cells in lacunae in hyaline matrix), (2) fibrocartilage (rounded cells in lacunae in fibrous matrix), (3) fibrous tissue (elongated cells in fibrous tissue), (4) bone, (5) scaffold material, and (6) others. The ability to discriminate between the tissue types was determined using conventional or polarized light microscopy, and the interobserver variability was evaluated. Results We describe the application of the stereological method. In the example, we assessed the defect repair tissue volume to be 4.4 mm3 (CE = 0.01). The tissue fractions were subsequently evaluated. Polarized light illumination of the slides improved discrimination between hyaline cartilage and fibrocartilage and increased the interobserver agreement compared with conventional transmitted light. Conclusion We have applied a design-unbiased method for quantitative evaluation of cartilage repair, and we propose this algorithm as a natural supplement to existing descriptive semiquantitative scoring systems. We also propose that polarized light is effective for discrimination between hyaline cartilage and fibrocartilage. PMID:26069715

Sampling methods for rumen microbial counts by Real-Time PCR techniques

Directory of Open Access Journals (Sweden)

S. Puppo

2010-02-01

Full Text Available Fresh rumen samples were withdrawn from 4 cannulated buffalo females fed a fibrous diets in order to quantify bacteria concentration in the rumen by Real-Time PCR techniques. To obtain DNA of a good quality from whole rumen fluid, eight (M1-M8 different pre-filtration methods (cheese cloths, glass-fibre and nylon filter in combination with various centrifugation speeds (1000, 5000 and 14,000 rpm were tested. Genomic DNA extraction was performed either on fresh or frozen samples (-20°C. The quantitative bacteria analysis was realized according to Real-Time PCR procedure for Butyrivibrio fibrisolvens reported in literature. M5 resulted the best sampling procedure allowing to obtain a suitable genomic DNA. No differences were revealed between fresh and frozen samples.

Real-time three-dimensional temperature mapping in photothermal therapy with optoacoustic tomography

Science.gov (United States)

Oyaga Landa, Francisco Javier; Deán-Ben, Xosé Luís.; Sroka, Ronald; Razansky, Daniel

2017-07-01

Ablation and photothermal therapy are widely employed medical protocols where the selective destruction of tissue is a necessity as in cancerous tissue removal or vascular and brain abnormalities. Tissue denaturation takes place when the temperature reaches a threshold value while the time of exposure determines the lesion size. Therefore, the spatio-temporal distribution of temperature plays a crucial role in the outcome of these clinical interventions. We demonstrate fast volumetric temperature mapping with optoacoustic tomography based on real-time optoacoustic readings from the treated region. The performance of the method was investigated in tissue-mimicking phantom experiments. The new ability to non-invasively measure temperature volumetrically in an entire treated region with high spatial and temporal resolutions holds potential for improving safety and efficacy of thermal ablation and to advance the general applicability of laser-based therapy.

Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions.

Science.gov (United States)

Wu, Qingzhong; Prager, Katherine C; Goldstein, Tracey; Alt, David P; Galloway, Renee L; Zuerner, Richard L; Lloyd-Smith, James O; Schwacke, Lori

2014-08-11

Several real-time PCR assays are currently used for detection of pathogenic Leptospira spp.; however, few methods have been described for the successful evaluation of clinical urine samples. This study reports a rapid assay for the detection of pathogenic Leptospira spp. in California sea lions Zalophus californianus using real-time PCR with primers and a probe targeting the lipL32 gene. The PCR assay had high analytic sensitivity-the limit of detection was 3 genome copies per PCR volume using L. interrogans serovar Pomona DNA and 100% analytic specificity; it detected all pathogenic leptospiral serovars tested and none of the non-pathogenic Leptospira species (L. biflexa and L. meyeri serovar Semaranga), the intermediate species L. inadai, or the non-Leptospira pathogens tested. Our assay had an amplification efficiency of 1.00. Comparisons between the real-time PCR assay and culture isolation for detection of pathogenic Leptospira spp. in urine and kidney tissue samples from California sea lions showed that samples were more often positive by real-time PCR than by culture methods. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that our real-time PCR assay has high analytic sensitivity and specificity for the rapid detection of pathogenic Leptospira species in urine and kidney tissue samples.

Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR

Directory of Open Access Journals (Sweden)

López-Revilla Rubén

2010-05-01

Full Text Available Abstract Background We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR in the presence of the DNA intercalating agent EvaGreen. Results Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp-long insert of the human papillomavirus type 16 (HPV16 E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 × 102-2.5 × 106 initial pHV101 copies had threshold cycle (Ct values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5°C. Conclusions Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6-HPV16 copy per cell.

A real-time architecture for time-aware agents.

Science.gov (United States)

Prouskas, Konstantinos-Vassileios; Pitt, Jeremy V

2004-06-01

This paper describes the specification and implementation of a new three-layer time-aware agent architecture. This architecture is designed for applications and environments where societies of humans and agents play equally active roles, but interact and operate in completely different time frames. The architecture consists of three layers: the April real-time run-time (ART) layer, the time aware layer (TAL), and the application agents layer (AAL). The ART layer forms the underlying real-time agent platform. An original online, real-time, dynamic priority-based scheduling algorithm is described for scheduling the computation time of agent processes, and it is shown that the algorithm's O(n) complexity and scalable performance are sufficient for application in real-time domains. The TAL layer forms an abstraction layer through which human and agent interactions are temporally unified, that is, handled in a common way irrespective of their temporal representation and scale. A novel O(n2) interaction scheduling algorithm is described for predicting and guaranteeing interactions' initiation and completion times. The time-aware predicting component of a workflow management system is also presented as an instance of the AAL layer. The described time-aware architecture addresses two key challenges in enabling agents to be effectively configured and applied in environments where humans and agents play equally active roles. It provides flexibility and adaptability in its real-time mechanisms while placing them under direct agent control, and it temporally unifies human and agent interactions.

Coalescence measurements for evolving foams monitored by real-time projection imaging

International Nuclear Information System (INIS)

Myagotin, A; Helfen, L; Baumbach, T

2009-01-01

Real-time radiographic projection imaging together with novel spatio-temporal image analysis is presented to be a powerful technique for the quantitative analysis of coalescence processes accompanying the generation and temporal evolution of foams and emulsions. Coalescence events can be identified as discontinuities in a spatio-temporal image representing a sequence of projection images. Detection, identification of intensity and localization of the discontinuities exploit a violation criterion of the Fourier shift theorem and are based on recursive spatio-temporal image partitioning. The proposed method is suited for automated measurements of discontinuity rates (i.e., discontinuity intensity per unit time), so that large series of radiographs can be analyzed without user intervention. The application potential is demonstrated by the quantification of coalescence during the formation and decay of metal foams monitored by real-time x-ray radiography

Phase-shifting Real-time Holographic Microscopy applied in micro-structures surface analysis

International Nuclear Information System (INIS)

Brito, I V; Gesualdi, M R R; Muramatsu, M; Ricardo, J

2011-01-01

The microscopic real-time analysis of micro structured materials is of great importance in various domains of science and technology. For other hand, the holographic interferometry comprises a group of powerful optical methods for non-destructive testing in surface analysis. The holographic microscopy uses the holographic interferometric techniques to obtain quantitative intensity and phase information of the optical waves by microscopic systems. With the development of CCD cameras, computers (hardware and software), and new materials for holographic recording, these techniques can be used to replace the classical form of registration and became promising tools in surface analysis. In this work, we developed a prototype of Photorefractive and Digital Holographic Microscope for real-time analysis of micro-structured systems based on the phase-shifting real-time holographic interferometry techniques. Using this apparatus, we are made analysis of shapes and surfaces to obtain the phase maps and the 3D profiles of some samples.

Towards Real-Time Argumentation

Directory of Open Access Journals (Sweden)

Vicente JULIÁN

2016-07-01

Full Text Available In this paper, we deal with the problem of real-time coordination with the more general approach of reaching real-time agreements in MAS. Concretely, this work proposes a real-time argumentation framework in an attempt to provide agents with the ability of engaging in argumentative dialogues and come with a solution for their underlying agreement process within a bounded period of time. The framework has been implemented and evaluated in the domain of a customer support application. Concretely, we consider a society of agents that act on behalf of a group of technicians that must solve problems in a Technology Management Centre (TMC within a bounded time. This centre controls every process implicated in the provision of technological and customer support services to private or public organisations by means of a call centre. The contract signed between the TCM and the customer establishes penalties if the specified time is exceeded.

Effect of ionizing radiation on the quantitative detection of Salmonella using real-time PCR

Energy Technology Data Exchange (ETDEWEB)

Lim, Sangyong; Jung, Jinwoo [Radiation Research Center for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Kim, Minjeong; Ryu, Sangryeol [Department of Food and Animal Biotechnology, School of Agricultural Biotechnology, Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921 (Korea, Republic of); Kim, Dongho [Radiation Research Center for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of)], E-mail: fungikim@kaeri.re.kr

2008-09-15

Food irradiation is an economically viable technology for inactivating foodborne pathogens, but irradiation can mask pathogens in unhygienically prepared food. The aim of this study was to investigate the effect of irradiation treatment on the detection of Salmonella using real-time PCR. Three commercially available kits were tested, of which the InstaGene Matrix procedure was most effective in preparing template DNA from Salmonella exposed to radiation in broth culture. The minimum level of detection by real-time PCR combined with InstaGene Matrix was 3 log units of Salmonella per milliliter. However, when pure cultures of Salmonella were irradiated at 3 and 5 kGy, the cycle threshold (C{sub T}) increased 1-1.5-fold compared to irradiation at 0 and 1 kGy. This indicated that irradiation treatment may result in an underestimation of bacterial counts due to radiation-induced DNA lesions. We also compared C{sub T} values in inoculated chicken homogenates before and after irradiation, which in this model caused a 1.3-3.3-fold underestimation of bacterial counts with respect to irradiation dose.

Real time programming environment for Windows

Energy Technology Data Exchange (ETDEWEB)

LaBelle, D.R. [LaBelle (Dennis R.), Clifton Park, NY (United States)

1998-04-01

This document provides a description of the Real Time Programming Environment (RTProE). RTProE tools allow a programmer to create soft real time projects under general, multi-purpose operating systems. The basic features necessary for real time applications are provided by RTProE, leaving the programmer free to concentrate efforts on his specific project. The current version supports Microsoft Windows{trademark} 95 and NT. The tasks of real time synchronization and communication with other programs are handled by RTProE. RTProE includes a generic method for connecting a graphical user interface (GUI) to allow real time control and interaction with the programmer`s product. Topics covered in this paper include real time performance issues, portability, details of shared memory management, code scheduling, application control, Operating System specific concerns and the use of Computer Aided Software Engineering (CASE) tools. The development of RTProE is an important step in the expansion of the real time programming community. The financial costs associated with using the system are minimal. All source code for RTProE has been made publicly available. Any person with access to a personal computer, Windows 95 or NT, and C or FORTRAN compilers can quickly enter the world of real time modeling and simulation.

Relationship Between Ebola Virus Real-Time Quantitative Polymerase Chain Reaction-Based Threshold Cycle Value and Virus Isolation From Human Plasma.

Science.gov (United States)

Spengler, Jessica R; McElroy, Anita K; Harmon, Jessica R; Ströher, Ute; Nichol, Stuart T; Spiropoulou, Christina F

2015-10-01

We performed a longitudinal analysis of plasma samples obtained from 4 patients with Ebola virus (EBOV) disease (EVD) to determine the relationship between the real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)-based threshold cycle (Ct) value and the presence of infectious EBOV. EBOV was not isolated from plasma samples with a Ct value of >35.5 or >12 days after onset of symptoms. EBOV was not isolated from plasma samples in which anti-EBOV nucleoprotein immunoglobulin G was detected. These data demonstrate the utility of interpreting qRT-PCR results in the context of the course of EBOV infection and associated serological responses for patient-management decisions. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus.

Science.gov (United States)

Guionie, O; Toquin, D; Sellal, E; Bouley, S; Zwingelstein, F; Allée, C; Bougeard, S; Lemière, S; Eterradossi, N

2007-02-01

Avian metapneumovirus (AMPV) is an important pathogen causing respiratory diseases and egg drops in several avian species. Four AMPV subgroups have been identified. The laboratory diagnosis of AMPV infections relies on serological methods, on labour-intensive virus isolation procedures, and on recently developed subgroup specific reverse transcription PCR (RT-PCR) protocols. In the present study, both the specificity and sensitivity of a commercial real-time reverse transcription PCR (RRT-PCR) for the detection and identification of the four AMPV subgroups were evaluated. Fifteen non-AMPV avian viruses belonging to 7 genera and 32 AMPV belonging to the 4 subgroups were tested. No non-AMPV virus was detected, whereas all AMPV viruses were identified in agreement with their previous molecular and antigenic subgroup assignment. The sensitivity and quantitating ability of the RRT-PCR assay were determined using serial dilutions of RNA derived either from AMPV virus stocks or from runoff transcripts. In all cases, linear dose/responses were observed. The detection limits of the different subgroups ranged from 500 to 5000 RNA copies and from 0.03 to 3.16TCID50/ml. The results were reproducible under laboratory conditions, thus showing that quantitative RRT-PCR is a new and powerful tool for the rapid and sensitive detection, identification and quantitation of AMPVs.

An In-Home Digital Network Architecture for Real-Time and Non-Real-Time Communication

NARCIS (Netherlands)

Scholten, Johan; Jansen, P.G.; Hanssen, F.T.Y.; Hattink, Tjalling

2002-01-01

This paper describes an in-home digital network architecture that supports both real-time and non-real-time communication. The architecture deploys a distributed token mechanism to schedule communication streams and to offer guaranteed quality-ofservice. Essentially, the token mechanism prevents

Development and validation of real-time simulation of X-ray imaging with respiratory motion.

Science.gov (United States)

Vidal, Franck P; Villard, Pierre-Frédéric

2016-04-01

We present a framework that combines evolutionary optimisation, soft tissue modelling and ray tracing on GPU to simultaneously compute the respiratory motion and X-ray imaging in real-time. Our aim is to provide validated building blocks with high fidelity to closely match both the human physiology and the physics of X-rays. A CPU-based set of algorithms is presented to model organ behaviours during respiration. Soft tissue deformation is computed with an extension of the Chain Mail method. Rigid elements move according to kinematic laws. A GPU-based surface rendering method is proposed to compute the X-ray image using the Beer-Lambert law. It is provided as an open-source library. A quantitative validation study is provided to objectively assess the accuracy of both components: (i) the respiration against anatomical data, and (ii) the X-ray against the Beer-Lambert law and the results of Monte Carlo simulations. Our implementation can be used in various applications, such as interactive medical virtual environment to train percutaneous transhepatic cholangiography in interventional radiology, 2D/3D registration, computation of digitally reconstructed radiograph, simulation of 4D sinograms to test tomography reconstruction tools. Copyright © 2015 Elsevier Ltd. All rights reserved.

MARTe: A Multiplatform Real-Time Framework

Science.gov (United States)

Neto, André C.; Sartori, Filippo; Piccolo, Fabio; Vitelli, Riccardo; De Tommasi, Gianmaria; Zabeo, Luca; Barbalace, Antonio; Fernandes, Horacio; Valcarcel, Daniel F.; Batista, Antonio J. N.

2010-04-01

Development of real-time applications is usually associated with nonportable code targeted at specific real-time operating systems. The boundary between hardware drivers, system services, and user code is commonly not well defined, making the development in the target host significantly difficult. The Multithreaded Application Real-Time executor (MARTe) is a framework built over a multiplatform library that allows the execution of the same code in different operating systems. The framework provides the high-level interfaces with hardware, external configuration programs, and user interfaces, assuring at the same time hard real-time performances. End-users of the framework are required to define and implement algorithms inside a well-defined block of software, named Generic Application Module (GAM), that is executed by the real-time scheduler. Each GAM is reconfigurable with a set of predefined configuration meta-parameters and interchanges information using a set of data pipes that are provided as inputs and required as output. Using these connections, different GAMs can be chained either in series or parallel. GAMs can be developed and debugged in a non-real-time system and, only once the robustness of the code and correctness of the algorithm are verified, deployed to the real-time system. The software also supplies a large set of utilities that greatly ease the interaction and debugging of a running system. Among the most useful are a highly efficient real-time logger, HTTP introspection of real-time objects, and HTTP remote configuration. MARTe is currently being used to successfully drive the plasma vertical stabilization controller on the largest magnetic confinement fusion device in the world, with a control loop cycle of 50 ?s and a jitter under 1 ?s. In this particular project, MARTe is used with the Real-Time Application Interface (RTAI)/Linux operating system exploiting the new ?86 multicore processors technology.

Detection and enumeration of Salmonella enteritidis in homemade ice cream associated with an outbreak: comparison of conventional and real-time PCR methods.

Science.gov (United States)

Seo, K H; Valentin-Bon, I E; Brackett, R E

2006-03-01

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.

Real-Time and Real-Fast Performance of General-Purpose and Real-Time Operating Systems in Multithreaded Physical Simulation of Complex Mechanical Systems

Directory of Open Access Journals (Sweden)

Carlos Garre

2014-01-01

Full Text Available Physical simulation is a valuable tool in many fields of engineering for the tasks of design, prototyping, and testing. General-purpose operating systems (GPOS are designed for real-fast tasks, such as offline simulation of complex physical models that should finish as soon as possible. Interfacing hardware at a given rate (as in a hardware-in-the-loop test requires instead maximizing time determinism, for which real-time operating systems (RTOS are designed. In this paper, real-fast and real-time performance of RTOS and GPOS are compared when simulating models of high complexity with large time steps. This type of applications is usually present in the automotive industry and requires a good trade-off between real-fast and real-time performance. The performance of an RTOS and a GPOS is compared by running a tire model scalable on the number of degrees-of-freedom and parallel threads. The benchmark shows that the GPOS present better performance in real-fast runs but worse in real-time due to nonexplicit task switches and to the latency associated with interprocess communication (IPC and task switch.

GNSS global real-time augmentation positioning: Real-time precise satellite clock estimation, prototype system construction and performance analysis

Science.gov (United States)

Chen, Liang; Zhao, Qile; Hu, Zhigang; Jiang, Xinyuan; Geng, Changjiang; Ge, Maorong; Shi, Chuang

2018-01-01

Lots of ambiguities in un-differenced (UD) model lead to lower calculation efficiency, which isn't appropriate for the high-frequency real-time GNSS clock estimation, like 1 Hz. Mixed differenced model fusing UD pseudo-range and epoch-differenced (ED) phase observations has been introduced into real-time clock estimation. In this contribution, we extend the mixed differenced model for realizing multi-GNSS real-time clock high-frequency updating and a rigorous comparison and analysis on same conditions are performed to achieve the best real-time clock estimation performance taking the efficiency, accuracy, consistency and reliability into consideration. Based on the multi-GNSS real-time data streams provided by multi-GNSS Experiment (MGEX) and Wuhan University, GPS + BeiDou + Galileo global real-time augmentation positioning prototype system is designed and constructed, including real-time precise orbit determination, real-time precise clock estimation, real-time Precise Point Positioning (RT-PPP) and real-time Standard Point Positioning (RT-SPP). The statistical analysis of the 6 h-predicted real-time orbits shows that the root mean square (RMS) in radial direction is about 1-5 cm for GPS, Beidou MEO and Galileo satellites and about 10 cm for Beidou GEO and IGSO satellites. Using the mixed differenced estimation model, the prototype system can realize high-efficient real-time satellite absolute clock estimation with no constant clock-bias and can be used for high-frequency augmentation message updating (such as 1 Hz). The real-time augmentation message signal-in-space ranging error (SISRE), a comprehensive accuracy of orbit and clock and effecting the users' actual positioning performance, is introduced to evaluate and analyze the performance of GPS + BeiDou + Galileo global real-time augmentation positioning system. The statistical analysis of real-time augmentation message SISRE is about 4-7 cm for GPS, whlile 10 cm for Beidou IGSO/MEO, Galileo and about 30 cm

Portable Dew Point Mass Spectrometry System for Real-Time Gas and Moisture Analysis

Science.gov (United States)

Arkin, C.; Gillespie, Stacey; Ratzel, Christopher

2010-01-01

A portable instrument incorporates both mass spectrometry and dew point measurement to provide real-time, quantitative gas measurements of helium, nitrogen, oxygen, argon, and carbon dioxide, along with real-time, quantitative moisture analysis. The Portable Dew Point Mass Spectrometry (PDP-MS) system comprises a single quadrupole mass spectrometer and a high vacuum system consisting of a turbopump and a diaphragm-backing pump. A capacitive membrane dew point sensor was placed upstream of the MS, but still within the pressure-flow control pneumatic region. Pressure-flow control was achieved with an upstream precision metering valve, a capacitance diaphragm gauge, and a downstream mass flow controller. User configurable LabVIEW software was developed to provide real-time concentration data for the MS, dew point monitor, and sample delivery system pressure control, pressure and flow monitoring, and recording. The system has been designed to include in situ, NIST-traceable calibration. Certain sample tubing retains sufficient water that even if the sample is dry, the sample tube will desorb water to an amount resulting in moisture concentration errors up to 500 ppm for as long as 10 minutes. It was determined that Bev-A-Line IV was the best sample line to use. As a result of this issue, it is prudent to add a high-level humidity sensor to PDP-MS so such events can be prevented in the future.

The numbers game: quantitative analysis of Neorickettsia sp. propagation through complex life cycle of its digenean host using real-time qPCR.

Science.gov (United States)

Greiman, Stephen E; Tkach, Vasyl V

2016-07-01

Bacteria of the genus Neorickettsia are obligate intracellular endosymbionts of parasitic flukes (Digenea) and are passed through the entire complex life cycle of the parasite by vertical transmission. Several species of Neorickettsia are known to cause diseases in domestic animals, wildlife, and humans. Quantitative data on the transmission of the bacteria through the digenean life cycle is almost completely lacking. This study quantified for the first time the abundance of Neorickettsia within multiple stages of the life cycle of the digenean Plagiorchis elegans. Snails Lymnaea stagnalis collected from a pond in North Dakota were screened for the presence of digenean cercariae, which were subsequently tested for the presence of Neorickettsia. Three L. stagnalis were found shedding P. elegans cercariae infected with Neorickettsia. These snails were used to initiate three separate laboratory life cycles and obtain all life cycle stages for bacterial quantification. A quantitative real-time PCR assay targeting the GroEL gene was developed to enumerate Neorickettsia sp. within different stages of the digenean life cycle. The number of bacteria significantly increased throughout all stages, from eggs to adults. The two largest increases in number of bacteria occurred during the period from eggs to cercariae and from 6-day metacercariae to 48-h juvenile worms. These two periods seem to be the most important for Neorickettsia propagation through the complex digenean life cycle and maturation in the definitive host.

Single-cell real-time imaging of transgene expression upon lipofection

Energy Technology Data Exchange (ETDEWEB)

Fiume, Giuseppe [Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa (Italy); Di Rienzo, Carmine [Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa (Italy); NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127, Pisa (Italy); Marchetti, Laura [Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa (Italy); Pozzi, Daniela; Caracciolo, Giulio [Department of Molecular Medicine, “Sapienza” University of Rome, Viale Regina Elena 291, 00161, Rome (Italy); Cardarelli, Francesco, E-mail: francesco.cardarelli@iit.it [Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa (Italy)

2016-05-20

Here we address the process of lipofection by quantifying the expression of a genetically-encoded fluorescent reporter at the single-cell level, and in real-time, by confocal imaging in live cells. The Lipofectamine gold-standard formulation is compared to the alternative promising DC-Chol/DOPE formulation. In both cases, we report that only dividing cells are able to produce a detectable amount of the fluorescent reporter protein. Notably, by measuring fluorescence over time in each pair of daughter cells, we find that Lipofectamine-based transfection statistically yields a remarkably higher degree of “symmetry” in protein expression between daughter cells as compared to DC-Chol/DOPE. A model is envisioned in which the degree of symmetry of protein expression is linked to the number of bioavailable DNA copies within the cell before nuclear breakdown. Reported results open new perspectives for the understanding of the lipofection mechanism and define a new experimental platform for the quantitative comparison of transfection reagents. -- Highlights: •The process of lipofection is followed by quantifying the transgene expression in real time. •The Lipofectamine gold-standard is compared to the promising DC-Chol/DOPE formulation. •We report that only dividing cells are able to produce the fluorescent reporter protein. •The degree of symmetry of protein expression in daughter cells is linked to DNA bioavailability. •A new experimental platform for the quantitative comparison of transfection reagents is proposed.

Single-cell real-time imaging of transgene expression upon lipofection

International Nuclear Information System (INIS)

Fiume, Giuseppe; Di Rienzo, Carmine; Marchetti, Laura; Pozzi, Daniela; Caracciolo, Giulio; Cardarelli, Francesco

2016-01-01

Here we address the process of lipofection by quantifying the expression of a genetically-encoded fluorescent reporter at the single-cell level, and in real-time, by confocal imaging in live cells. The Lipofectamine gold-standard formulation is compared to the alternative promising DC-Chol/DOPE formulation. In both cases, we report that only dividing cells are able to produce a detectable amount of the fluorescent reporter protein. Notably, by measuring fluorescence over time in each pair of daughter cells, we find that Lipofectamine-based transfection statistically yields a remarkably higher degree of “symmetry” in protein expression between daughter cells as compared to DC-Chol/DOPE. A model is envisioned in which the degree of symmetry of protein expression is linked to the number of bioavailable DNA copies within the cell before nuclear breakdown. Reported results open new perspectives for the understanding of the lipofection mechanism and define a new experimental platform for the quantitative comparison of transfection reagents. -- Highlights: •The process of lipofection is followed by quantifying the transgene expression in real time. •The Lipofectamine gold-standard is compared to the promising DC-Chol/DOPE formulation. •We report that only dividing cells are able to produce the fluorescent reporter protein. •The degree of symmetry of protein expression in daughter cells is linked to DNA bioavailability. •A new experimental platform for the quantitative comparison of transfection reagents is proposed.

Scalable Real-Time Negotiation Toolkit

National Research Council Canada - National Science Library

Lesser, Victor

2004-01-01

... to implement an adaptive distributed sensor network. These activities involved the development of a distributed soft, real-time heuristic resource allocation protocol, the development of a domain-independent soft, real time agent architecture...

Development and validation of a real-time PCR assay for specific and sensitive detection of canid herpesvirus 1.

Science.gov (United States)

Decaro, Nicola; Amorisco, Francesca; Desario, Costantina; Lorusso, Eleonora; Camero, Michele; Bellacicco, Anna Lucia; Sciarretta, Rossana; Lucente, Maria Stella; Martella, Vito; Buonavoglia, Canio

2010-10-01

A TaqMan-based real-time PCR assay targeting the glycoprotein B-encoding gene was developed for diagnosis of canid herpesvirus 1 (CHV-1) infection. The established assay was highly specific, since no cross-reactions were observed with other canine DNA viruses, including canine parvovirus type 2, canine minute virus, or canine adenovirus types 1 and 2. The detection limit was 10(1) and 1.20 x 10(1) DNA copies per 10 microl(-1) of template for standard DNA and a CHV-1-positive kidney sample, respectively: about 1-log higher than a gel-based PCR assay targeting the thymidine kinase gene. The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. CHV-1 isolates of different geographical origins were recognised by the TaqMan assay. Tissues and clinical samples collected from three pups which died of CHV-1 neonatal infection were also tested, displaying a wide distribution of CHV-l DNA in their organs. Unlike other CHV-1-specific diagnostic methods, this quantitative assay permits simultaneous detection and quantitation of CHV-1 DNA in a wide range of canine tissues and body fluids, thus providing a useful tool for confirmation of a clinical diagnosis, for the study of viral pathogenesis and for evaluation of the efficacy of vaccines and antiviral drugs. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses

DEFF Research Database (Denmark)

Stilwell, Natalie K.; Whittington, Richard J.; Hick, Paul M.

2018-01-01

Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ran...

Quantitative breast tissue characterization using grating-based x-ray phase-contrast imaging

Science.gov (United States)

Willner, M.; Herzen, J.; Grandl, S.; Auweter, S.; Mayr, D.; Hipp, A.; Chabior, M.; Sarapata, A.; Achterhold, K.; Zanette, I.; Weitkamp, T.; Sztrókay, A.; Hellerhoff, K.; Reiser, M.; Pfeiffer, F.

2014-04-01

X-ray phase-contrast imaging has received growing interest in recent years due to its high capability in visualizing soft tissue. Breast imaging became the focus of particular attention as it is considered the most promising candidate for a first clinical application of this contrast modality. In this study, we investigate quantitative breast tissue characterization using grating-based phase-contrast computed tomography (CT) at conventional polychromatic x-ray sources. Different breast specimens have been scanned at a laboratory phase-contrast imaging setup and were correlated to histopathology. Ascertained tumor types include phylloides tumor, fibroadenoma and infiltrating lobular carcinoma. Identified tissue types comprising adipose, fibroglandular and tumor tissue have been analyzed in terms of phase-contrast Hounsfield units and are compared to high-quality, high-resolution data obtained with monochromatic synchrotron radiation, as well as calculated values based on tabulated tissue properties. The results give a good impression of the method’s prospects and limitations for potential tumor detection and the associated demands on such a phase-contrast breast CT system. Furthermore, the evaluated quantitative tissue values serve as a reference for simulations and the design of dedicated phantoms for phase-contrast mammography.

Growing tissues in real and simulated microgravity: new methods for tissue engineering.

Science.gov (United States)

Grimm, Daniela; Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Ulbrich, Claudia; Magnusson, Nils E; Infanger, Manfred; Bauer, Johann

2014-12-01

Tissue engineering in simulated (s-) and real microgravity (r-μg) is currently a topic in Space medicine contributing to biomedical sciences and their applications on Earth. The principal aim of this review is to highlight the advances and accomplishments in the field of tissue engineering that could be achieved by culturing cells in Space or by devices created to simulate microgravity on Earth. Understanding the biology of three-dimensional (3D) multicellular structures is very important for a more complete appreciation of in vivo tissue function and advancing in vitro tissue engineering efforts. Various cells exposed to r-μg in Space or to s-μg created by a random positioning machine, a 2D-clinostat, or a rotating wall vessel bioreactor grew in the form of 3D tissues. Hence, these methods represent a new strategy for tissue engineering of a variety of tissues, such as regenerated cartilage, artificial vessel constructs, and other organ tissues as well as multicellular cancer spheroids. These aggregates are used to study molecular mechanisms involved in angiogenesis, cancer development, and biology and for pharmacological testing of, for example, chemotherapeutic drugs or inhibitors of neoangiogenesis. Moreover, they are useful for studying multicellular responses in toxicology and radiation biology, or for performing coculture experiments. The future will show whether these tissue-engineered constructs can be used for medical transplantations. Unveiling the mechanisms of microgravity-dependent molecular and cellular changes is an up-to-date requirement for improving Space medicine and developing new treatment strategies that can be translated to in vivo models while reducing the use of laboratory animals.

Model Checking Real-Time Systems

DEFF Research Database (Denmark)

Bouyer, Patricia; Fahrenberg, Uli; Larsen, Kim Guldstrand

2018-01-01

This chapter surveys timed automata as a formalism for model checking real-time systems. We begin with introducing the model, as an extension of finite-state automata with real-valued variables for measuring time. We then present the main model-checking results in this framework, and give a hint...

Modular specification of real-time systems

DEFF Research Database (Denmark)

Inal, Recep

1994-01-01

Duration Calculus, a real-time interval logic, has been embedded in the Z specification language to provide a notation for real-time systems that combines the modularisation and abstraction facilities of Z with a logic suitable for reasoning about real-time properties. In this article the notation...

Hard Real-Time Networking on Firewire

NARCIS (Netherlands)

Zhang, Yuchen; Orlic, Bojan; Visser, Peter; Broenink, Jan

2005-01-01

This paper investigates the possibility of using standard, low-cost, widely used FireWire as a new generation fieldbus medium for real-time distributed control applications. A real-time software subsys- tem, RT-FireWire was designed that can, in combination with Linux-based real-time operating

A real-time fluorescent sensor specific to Mg2+: crystallographic evidence, DFT calculation and its use for quantitative determination of magnesium in drinking water.

Science.gov (United States)

Men, Guangwen; Chen, Chunrong; Zhang, Shitong; Liang, Chunshuang; Wang, Ying; Deng, Mengyu; Shang, Hongxing; Yang, Bing; Jiang, Shimei

2015-02-14

An "off-the-shelf" fluorescence "turn-on" Mg(2+) chemosensor 3,5-dichlorosalicylaldehyde (BCSA) was rationally designed and developed. This proposed sensor works based on Mg(2+)-induced formation of the 2 : 1 BCSA-Mg(2+) complex. The coordination of BSCA to Mg(2+) increases its structural rigidity generating a chelation-enhanced fluorescence (CHEF) effect which was confirmed by single crystal XRD studies of the BSCA-Mg(2+) complex and TD/DFT calculations. This sensor exhibits high sensitivity and selectivity for the quantitative monitoring of Mg(2+) with a wide detection range (0-40 μM), a low detection limit (2.89 × 10(-7) mol L(-1)) and a short response time (sensor can be utilized to monitor Mg(2+) in real time within actual samples from drinking water.

Multiprocessor scheduling for real-time systems

CERN Document Server

Baruah, Sanjoy; Buttazzo, Giorgio

2015-01-01

This book provides a comprehensive overview of both theoretical and pragmatic aspects of resource-allocation and scheduling in multiprocessor and multicore hard-real-time systems.  The authors derive new, abstract models of real-time tasks that capture accurately the salient features of real application systems that are to be implemented on multiprocessor platforms, and identify rules for mapping application systems onto the most appropriate models.  New run-time multiprocessor scheduling algorithms are presented, which are demonstrably better than those currently used, both in terms of run-time efficiency and tractability of off-line analysis.  Readers will benefit from a new design and analysis framework for multiprocessor real-time systems, which will translate into a significantly enhanced ability to provide formally verified, safety-critical real-time systems at a significantly lower cost.

Application of propidium monoazide quantitative real-time PCR to quantify the viability of Lactobacillus delbrueckii ssp. bulgaricus.

Science.gov (United States)

Shao, Yuyu; Wang, Zhaoxia; Bao, Qiuhua; Zhang, Heping

2016-12-01

In this study, a combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) was used to develop a method to determine the viability of cells of Lactobacillus delbrueckii ssp. bulgaricus ND02 (L. bulgaricus) that may have entered into a viable but nonculturable state. This can happen due to its susceptibility to cold shock during lyophilization and storage. Propidium monoazide concentration, PMA incubation time, and light exposure time were optimized to fully exploit the PMA-qPCR approach to accurately assess the total number of living L. bulgaricus ND02. Although PMA has little influence on living cells, when concentrations of PMA were higher than 30μg/mL the number of PCR-positive living bacteria decreased from 10 6 to 10 5 cfu/mL in comparison with qPCR enumeration. Mixtures of living and dead cells were used as method verification samples for enumeration by PMA-qPCR, demonstrating that this method was feasible and effective for distinguishing living cells of L. bulgaricus when mixed with a known number of dead cells. We suggest that several conditions need to be studied further before PMA-qPCR methods can be accurately used to distinguish living from dead cells for enumeration under more realistic sampling situations. However, this research provides a rapid way to enumerate living cells of L. bulgaricus and could be used to optimize selection of cryoprotectants in the lyophilization process and develop technologies for high cell density cultivation and optimal freeze-drying processes. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

Science.gov (United States)

Fazelahi, Mansoureh; Kia, Vahid; Kaghazian, Hooman; Paryan, Mahdi

2017-11-26

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10 1 to 10 7 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

Detection and quantitation analysis of cocaine and metabolites in fixed liver tissue and formalin solutions.

Science.gov (United States)

Cingolani, Mariano; Cippitelli, Marcello; Froldi, Rino; Gambaro, Veniero; Tassoni, Giovanna

2004-01-01

This study reports the results of the detection and quantitation of cocaine and its metabolites in liver tissues fixed in formalin and in the formalin solutions in which the same tissues were fixed. Toxicological analyses were performed on formalin-fixed liver samples from four cases of death of cocaine abusers and on formalin solutions (10% buffered, pH 7) in which the samples were preserved. Analyses carried out at the time of autopsy on body fluids and tissues allowed identification of cocaine and the metabolite benzoylecgonine. Liver tissue samples were preserved in formalin solutions for four weeks before analysis. Results only showed the presence of benzoylecgonine in the studied materials. The mean levels of recovery of benzoylecgonine in fixed tissues were 12.31% in liver and 84.47% in formalin from liver. Results indicated that benzoylecgonine has good stability, even in biological specimens subjected to chemical fixation.

Quantitative t2 values predict time from symptom onset in acute stroke patients

DEFF Research Database (Denmark)

Siemonsen, Susanne; Mouridsen, Kim; Holst, Brigitte

2009-01-01

BACKGROUND AND PURPOSE: We hypothesize that in comparison to diffusion-weighted imaging, quantitative T2 values (qT2) are more directly related to water uptake in ischemic tissue, depending on time from symptom onset. We measured the increase of qT2 in the infarct core to quantify the correlation...

Prototyping real-time systems

OpenAIRE

Clynch, Gary

1994-01-01

The traditional software development paradigm, the waterfall life cycle model, is defective when used for developing real-time systems. This thesis puts forward an executable prototyping approach for the development of real-time systems. A prototyping system is proposed which uses ESML (Extended Systems Modelling Language) as a prototype specification language. The prototyping system advocates the translation of non-executable ESML specifications into executable LOOPN (Language of Object ...

Two quantitative real-time PCR assays for the detection of penaeid shrimp and blue crab, crustacean shellfish allergens.

Science.gov (United States)

Eischeid, Anne C; Kim, Bang-hyun; Kasko, Sasha M

2013-06-19

Food allergen detection methods must be able to specifically detect minute quantities of an allergenic food in a complex food matrix. One technique that can be used is real-time PCR. For the work described here, real-time PCR assays were developed to detect penaeid shrimp and blue crab, crustacean shellfish allergens. The method was tested using shrimp meat and crab meat spiked into several types of foods, including canned soups, deli foods, meat, seafood, and prepared seafood products. Foods were spiked with either shrimp or crab at levels ranging from 0.1 to 10⁶ parts per million (ppm) and analyzed either raw or cooked by a variety of methods. Real-time PCR data were used to generate linear standard curves, and assays were evaluated with respect to linear range and reaction efficiency. Results indicate that both assays performed well in a variety of food types. High reaction efficiencies were achieved across a linear range of 6-8 orders of magnitude. Limits of detection were generally between 0.1 and 1 ppm. Cooking methods used to simulate thermal processing of foods had little effect on assay performance. This work demonstrates that real-time PCR can be a valuable tool in the detection of crustacean shellfish.

Aspects of Quantitation in Mass Spectrometry Imaging Investigated on Cryo-Sections of Spiked Tissue Homogenates.

Science.gov (United States)

Hansen, Heidi Toft; Janfelt, Christian

2016-12-06

Internal standards have been introduced in quantitative mass spectrometry imaging in order to compensate for differences in intensities throughout an image caused by, for example, difference in ion suppression or analyte extraction efficiency. To test how well the internal standards compensate for differences in tissue types in, for example, whole-body imaging, a set of tissue homogenates of different tissue types (lung, liver, kidney, heart, and brain) from rabbit was spiked to the same concentration with the drug amitriptyline and imaged in the same experiment using isotope labeled amitriptyline as internal standard. The results showed, even after correction with internal standard, significantly lower intensities from brain and to some extent also lung tissue, differences which may be ascribed to binding of the drug to proteins or lipids as known from traditional bioanalysis. The differences, which for these results range approximately within a factor of 3 (but for other compounds in other tissues could be higher), underscore the importance of preparing the standard curve in the same matrix as the unknown sample whenever possible. In, for example, whole-body imaging where a diversity of tissue types are present, this variation across tissue types will therefore add to the overall uncertainty in quantitation. The tissue homogenates were also used in a characterization of various phenomena in quantitative MSI, such as to study how the signal depends of the thickness of the cryo-section, and to assess the accuracy of calibration by droplet deposition. For experiments on liver tissue, calibration by spiked tissue homogenates and droplet deposition was found to provide highly similar results and in both cases linearity with R 2 values of 0.99. In the process, a new method was developed for preparation of standard curves of spiked tissue homogenates, based on the drilling of holes in a block of frozen liver homogenate, providing easy cryo-slicing and good quantitative

Software Design Methods for Real-Time Systems

Science.gov (United States)

1989-12-01

This module describes the concepts and methods used in the software design of real time systems . It outlines the characteristics of real time systems , describes...the role of software design in real time system development, surveys and compares some software design methods for real - time systems , and

Real-time Pricing in Power Markets

DEFF Research Database (Denmark)

Boom, Anette; Schwenen, Sebastian

We examine welfare e ects of real-time pricing in electricity markets. Before stochastic energy demand is known, competitive retailers contract with nal consumers who exogenously do not have real-time meters. After demand is realized, two electricity generators compete in a uniform price auction...... to satisfy demand from retailers acting on behalf of subscribed customers and from consumers with real-time meters. Increasing the number of consumers on real-time pricing does not always increase welfare since risk-averse consumers dislike uncertain and high prices arising through market power...

Real-time Pricing in Power Markets

DEFF Research Database (Denmark)

Boom, Anette; Schwenen, Sebastian

We examine welfare eects of real-time pricing in electricity markets. Before stochastic energy demand is known, competitive retailers contract with nal consumers who exogenously do not have real-time meters. After demand is realized, two electricity generators compete in a uniform price auction...... to satisfy demand from retailers acting on behalf of subscribed customers and from consumers with real-time meters. Increasing the number of consumers on real-time pricing does not always increase welfare since risk-averse consumers dislike uncertain and high prices arising through market power...

Comparative validation using quantitative real-time PCR (qPCR and conventional PCR of bovine semen centrifuged in continuous density gradient

Directory of Open Access Journals (Sweden)

M.V. Resende

2011-06-01

Full Text Available The objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05. The percentage of female embryos in the Percoll and OptiPrep groups was 62.0% and 47.1%, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

Distributed, Embedded and Real-time Java Systems

CERN Document Server

Wellings, Andy

2012-01-01

Research on real-time Java technology has been prolific over the past decade, leading to a large number of corresponding hardware and software solutions, and frameworks for distributed and embedded real-time Java systems.  This book is aimed primarily at researchers in real-time embedded systems, particularly those who wish to understand the current state of the art in using Java in this domain.  Much of the work in real-time distributed, embedded and real-time Java has focused on the Real-time Specification for Java (RTSJ) as the underlying base technology, and consequently many of the Chapters in this book address issues with, or solve problems using, this framework. Describes innovative techniques in: scheduling, memory management, quality of service and communication systems supporting real-time Java applications; Includes coverage of multiprocessor embedded systems and parallel programming; Discusses state-of-the-art resource management for embedded systems, including Java’s real-time garbage collect...

The acellular matrix (ACM) for bladder tissue engineering: A quantitative magnetic resonance imaging study.

Science.gov (United States)

Cheng, Hai-Ling Margaret; Loai, Yasir; Beaumont, Marine; Farhat, Walid A

2010-08-01

Bladder acellular matrices (ACMs) derived from natural tissue are gaining increasing attention for their role in tissue engineering and regeneration. Unlike conventional scaffolds based on biodegradable polymers or gels, ACMs possess native biomechanical and many acquired biologic properties. Efforts to optimize ACM-based scaffolds are ongoing and would be greatly assisted by a noninvasive means to characterize scaffold properties and monitor interaction with cells. MRI is well suited to this role, but research with MRI for scaffold characterization has been limited. This study presents initial results from quantitative MRI measurements for bladder ACM characterization and investigates the effects of incorporating hyaluronic acid, a natural biomaterial useful in tissue-engineering and regeneration. Measured MR relaxation times (T(1), T(2)) and diffusion coefficient were consistent with increased water uptake and glycosaminoglycan content observed on biochemistry in hyaluronic acid ACMs. Multicomponent MRI provided greater specificity, with diffusion data showing an acellular environment and T(2) components distinguishing the separate effects of increased glycosaminoglycans and hydration. These results suggest that quantitative MRI may provide useful information on matrix composition and structure, which is valuable in guiding further development using bladder ACMs for organ regeneration and in strategies involving the use of hyaluronic acid.

Research of real-time communication software

Science.gov (United States)

Li, Maotang; Guo, Jingbo; Liu, Yuzhong; Li, Jiahong

2003-11-01

Real-time communication has been playing an increasingly important role in our work, life and ocean monitor. With the rapid progress of computer and communication technique as well as the miniaturization of communication system, it is needed to develop the adaptable and reliable real-time communication software in the ocean monitor system. This paper involves the real-time communication software research based on the point-to-point satellite intercommunication system. The object-oriented design method is adopted, which can transmit and receive video data and audio data as well as engineering data by satellite channel. In the real-time communication software, some software modules are developed, which can realize the point-to-point satellite intercommunication in the ocean monitor system. There are three advantages for the real-time communication software. One is that the real-time communication software increases the reliability of the point-to-point satellite intercommunication system working. Second is that some optional parameters are intercalated, which greatly increases the flexibility of the system working. Third is that some hardware is substituted by the real-time communication software, which not only decrease the expense of the system and promotes the miniaturization of communication system, but also aggrandizes the agility of the system.

Rapid diagnosis of sepsis with TaqMan-Based multiplex real-time PCR.

Science.gov (United States)

Liu, Chang-Feng; Shi, Xin-Ping; Chen, Yun; Jin, Ye; Zhang, Bing

2018-02-01

The survival rate of septic patients mainly depends on a rapid and reliable diagnosis. A rapid, broad range, specific and sensitive quantitative diagnostic test is the urgent need. Thus, we developed a TaqMan-Based Multiplex real-time PCR assays to identify bloodstream pathogens within a few hours. Primers and TaqMan probes were designed to be complementary to conserved regions in the 16S rDNA gene of different kinds of bacteria. To evaluate accurately, sensitively, and specifically, the known bacteria samples (Standard strains, whole blood samples) are determined by TaqMan-Based Multiplex real-time PCR. In addition, 30 blood samples taken from patients with clinical symptoms of sepsis were tested by TaqMan-Based Multiplex real-time PCR and blood culture. The mean frequency of positive for Multiplex real-time PCR was 96% at a concentration of 100 CFU/mL, and it was 100% at a concentration greater than 1000 CFU/mL. All the known blood samples and Standard strains were detected positively by TaqMan-Based Multiplex PCR, no PCR products were detected when DNAs from other bacterium were used in the multiplex assay. Among the 30 patients with clinical symptoms of sepsis, 18 patients were confirmed positive by Multiplex real-time PCR and seven patients were confirmed positive by blood culture. TaqMan-Based Multiplex real-time PCR assay with highly sensitivity, specificity and broad detection range, is a rapid and accurate method in the detection of bacterial pathogens of sepsis and should have a promising usage in the diagnosis of sepsis. © 2017 Wiley Periodicals, Inc.

Integration of MDSplus in real-time systems

International Nuclear Information System (INIS)

Luchetta, A.; Manduchi, G.; Taliercio, C.

2006-01-01

RFX-mod makes extensive usage of real-time systems for feedback control and uses MDSplus to interface them to the main Data Acquisition system. For this purpose, the core of MDSplus has been ported to VxWorks, the operating system used for real-time control in RFX. Using this approach, it is possible to integrate real-time systems, but MDSplus is used only for non-real-time tasks, i.e. those tasks which are executed before and after the pulse and whose performance does not affect the system time constraints. More extensive use of MDSplus in real-time systems is foreseen, and a real-time layer for MDSplus is under development, which will provide access to memory-mapped pulse files, shared by the tasks running on the same CPU. Real-time communication will also be integrated in the MDSplus core to provide support for distributed memory-mapped pulse files

Estimate of the real-time respiratory simulation system in cyberknife image-guided radiosurgery

International Nuclear Information System (INIS)

Min, Chul Kee; Chung, Weon Kuu; Lee, Suk

2010-01-01

The purpose of this study was to evaluate the target accuracy according to the movement with respiration of an actual patient in a quantitative way by developing a real-time respiratory simulation system (RRSS), including a patient customized 3D moving phantom. The real-time respiratory simulation system (RRSS) consists of two robots in order to implement both the movement of body surfaces and the movement of internal organs caused by respiration. The quantitative evaluation for the 3D movement of the RRSS was performed using a real-time laser displacement sensor for each axis. The average difference in the static movement of the RRSS was about 0.01 ∼ 0.06 mm. Also, in the evaluation of the dynamic movement by producing a formalized sine wave with the phase of four seconds per cycle, the difference between the measured and the calculated values for each cycle length in the robot that was in charge of body surfaces and the robot that was in charge of the movement of internal tumors showed 0.10 ∼ 0.55 seconds, and the correlation coefficients between the calculated and the measured values were 0.998 ∼ 0.999. The differences between the maximum and the minimum amplitudes were 0.01 ∼ 0.06 mm, and the reproducibility was within ±0.5 mm. In the case of the application and non-application of respiration, the target errors were -0.05 ∼ 1.05 mm and -0.13 ∼ 0.74 mm, respectively, and the entire target errors were 1.30 mm and 0.79 mm, respectively. Based on the accuracy in the RRSS system, various respiration patterns of patients can be reproduced in real-time. Also, this system can be used as an optimal tool for applying patient customized accuracy management in image-guided radiosurgery.

Estimate of the real-time respiratory simulation system in cyberknife image-guided radiosurgery

Energy Technology Data Exchange (ETDEWEB)

Min, Chul Kee [Konyang Univ. Hospital, Daejeon (Korea, Republic of); Kyonggi University, Seoul (Korea, Republic of); Chung, Weon Kuu [Konyang Univ. Hospital, Daejeon (Korea, Republic of); Lee, Suk [Korea University, Seoul (Korea, Republic of); and others

2010-01-15

The purpose of this study was to evaluate the target accuracy according to the movement with respiration of an actual patient in a quantitative way by developing a real-time respiratory simulation system (RRSS), including a patient customized 3D moving phantom. The real-time respiratory simulation system (RRSS) consists of two robots in order to implement both the movement of body surfaces and the movement of internal organs caused by respiration. The quantitative evaluation for the 3D movement of the RRSS was performed using a real-time laser displacement sensor for each axis. The average difference in the static movement of the RRSS was about 0.01 {approx} 0.06 mm. Also, in the evaluation of the dynamic movement by producing a formalized sine wave with the phase of four seconds per cycle, the difference between the measured and the calculated values for each cycle length in the robot that was in charge of body surfaces and the robot that was in charge of the movement of internal tumors showed 0.10 {approx} 0.55 seconds, and the correlation coefficients between the calculated and the measured values were 0.998 {approx} 0.999. The differences between the maximum and the minimum amplitudes were 0.01 {approx} 0.06 mm, and the reproducibility was within {+-}0.5 mm. In the case of the application and non-application of respiration, the target errors were -0.05 {approx} 1.05 mm and -0.13 {approx} 0.74 mm, respectively, and the entire target errors were 1.30 mm and 0.79 mm, respectively. Based on the accuracy in the RRSS system, various respiration patterns of patients can be reproduced in real-time. Also, this system can be used as an optimal tool for applying patient customized accuracy management in image-guided radiosurgery.

Selection and validation of reference genes for gene expression analysis in switchgrass (Panicum virgatum using quantitative real-time RT-PCR.

Directory of Open Access Journals (Sweden)

Jacinta Gimeno

Full Text Available Switchgrass (Panicum virgatum has received a lot of attention as a forage and bioenergy crop during the past few years. Gene expression studies are in progress to improve new traits and develop new cultivars. Quantitative real time PCR (qRT-PCR has emerged as an important technique to study gene expression analysis. For accurate and reliable results, normalization of data with reference genes is essential. In this work, we evaluate the stability of expression of genes to use as reference for qRT-PCR in the grass P. virgatum. Eleven candidate reference genes, including eEF-1α, UBQ6, ACT12, TUB6, eIF-4a, GAPDH, SAMDC, TUA6, CYP5, U2AF, and FTSH4, were validated for qRT-PCR normalization in different plant tissues and under different stress conditions. The expression stability of these genes was verified by the use of two distinct algorithms, geNorm and NormFinder. Differences were observed after comparison of the ranking of the candidate reference genes identified by both programs but eEF-1α, eIF-4a, CYP5 and U2AF are ranked as the most stable genes in the samples sets under study. Both programs discard the use of SAMDC and TUA6 for normalization. Validation of the reference genes proposed by geNorm and NormFinder were performed by normalization of transcript abundance of a group of target genes in different samples. Results show similar expression patterns when the best reference genes selected by both programs were used but differences were detected in the transcript abundance of the target genes. Based on the above research, we recommend the use of different statistical algorithms to identify the best reference genes for expression data normalization. The best genes selected in this study will help to improve the quality of gene expression data in a wide variety of samples in switchgrass.

High-Resolution Light Transmission Spectroscopy of Nanoparticles in Real Time

Science.gov (United States)

Tanner, Carol; Sun, Nan; Deatsch, Alison; Li, Frank; Ruggiero, Steven

2017-04-01

As implemented here, Light Transmission Spectroscopy (LTS) is a high-resolution real-time technique for eliminating spectral noise and systematic effects in wide band spectroscopic measurements of nanoparticles. In this work, we combine LTS with spectral inversion for the purpose of characterizing the size, shape, and number of nanoparticles in solution. The apparatus employs a wide-band multi-wavelength light source and grating spectrometers coupled to CCD detectors. The light source ranges from 210 to 2000 nm, and the wavelength dependent light detection system ranges from 200 to 1100 nm with model the total extinction cross-section, and spectral inversion is employed to obtain quantitative particle size distributions. Discussed are the precision, accuracy, resolution, and sensitivity of our results. The technique is quite versatile and can be applied to spectroscopic investigations where wideband, accurate, low-noise, real-time spectra are desired. University of Notre Dame Office of Research, College of Science, Department of Physics, and USDA.

Dense time discretization technique for verification of real time systems

International Nuclear Information System (INIS)

Makackas, Dalius; Miseviciene, Regina

2016-01-01

Verifying the real-time system there are two different models to control the time: discrete and dense time based models. This paper argues a novel verification technique, which calculates discrete time intervals from dense time in order to create all the system states that can be reached from the initial system state. The technique is designed for real-time systems specified by a piece-linear aggregate approach. Key words: real-time system, dense time, verification, model checking, piece-linear aggregate

Protein Analysis Using Real-Time PCR Instrumentation: Incorporation in an Integrated, Inquiry-Based Project

Science.gov (United States)

Southard, Jonathan N.

2014-01-01

Instrumentation for real-time PCR is used primarily for amplification and quantitation of nucleic acids. The capability to measure fluorescence while controlling temperature in multiple samples can also be applied to the analysis of proteins. Conformational stability and changes in stability due to ligand binding are easily assessed. Protein…

Quantitative Real-Time PCR Fecal Source Identification in the ...

Science.gov (United States)

Rivers in the Tillamook Basin play a vital role in supporting a thriving dairy and cheese-making industry, as well as providing a safe water resource for local human and wildlife populations. Historical concentrations of fecal bacteria in these waters are at times too high to allow for safe use leading to economic loss, endangerment of local wildlife, and poor conditions for recreational use. In this study, we employ host-associated qPCR methods for human (HF183/BacR287 and HumM2), ruminant (Rum2Bac), cattle (CowM2 and CowM3), canine (DG3 and DG37), and avian (GFD) fecal pollution combined with high-resolution geographic information system (GIS) land use data and general indicator bacteria measurements to elucidatewater quality spatial and temporal trends. Water samples (n=584) were collected over a 1-year period at 29 sites along the Trask, Kilchis, and Tillamook rivers and tributaries (Tillamook Basin, OR). A total of 16.6% of samples (n=97) yielded E. coli levels considered impaired based on Oregon Department of Environmental Quality bacteria criteria (406 MPN/100mL). Hostassociated genetic indicators were detected at frequencies of 39.2% (HF183/BacR287), 16.3% (HumM2), 74.6% (Rum2Bac), 13.0% (CowM2), 26.7% (CowM3), 19.8% (DG3), 3.2% (DG37), and 53.4% (GFD) across all water samples (n=584). Seasonal trends in avian, cattle, and human fecal pollution sources were evident over the study area. On a sample site basis, quantitative fecal source identification and

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

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Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

2014-12-01

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

Microsatellite instability typing in serum and tissue of patients with colorectal cancer: comparing real time PCR with hybridization probe and high-performance liquid chromatography.

Science.gov (United States)

Mokarram, P; Rismanchi, M; Alizadeh Naeeni, M; Mirab Samiee, S; Paryan, M; Alipour, A; Honardar, Z; Kavousipour, S; Naghibalhossaini, F; Mostafavi-Pour, Z; Monabati, A; Hosseni, S V; Shamsdin, S A

2014-05-01

Allelic variation of BAT-25 (a 25-repeat quasimonomorphic poly T) and BAT-26 (a 26-repeat quasimonomorphic polyA) loci as two mononucleotide microsatellite markers, were analyzed with high-performance liquid chromatography (HPLC) compared with Real-Time PCR using hybridization probes. BAT-26 and BAT-25 markers were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose microsatellite instability (MSI) status in patients with colorectal cancer (CRC). One of the pathways in colorectal tumor genesis is microsatellite instability (MSI+). MSI is detected in about 15% of all CRCs; 3% are of these are associated with Lynch syndrome and the other 12% are caused by sporadic. Colorectal tumors with MSI have distinctive features compared with microsatellite stable tumors. Due to the high percentage of MSI+ CRC in Iran, screening of this type of CRC is imperative. Two markers were analyzed in tissues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic CRC. The sensitivity and specificity of BAT-26 with real time PCR method (Hybridization probe) were 100% in comparison with sequencing method as the gold standard, while HPLC had a lower sensitivity and specificity. According to HPLC data, BAT-26 was more sensitive than BAT-25 in identifying MSI tumors. Therefore, MSI typing using the BAT-26 hybridization probe method compared to HPLC could be considered as an accurate method for diagnosing MSI in CRC tumors but not in serum circulating DNAs.

Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

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